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bioRxiv2020

Dissociating breathlessness symptoms from mood in asthma

Asthma is one of many chronic diseases in which discordance between objectively measured pathophysiology and symptom burden is well recognised. Understanding the influences on symptom burden beyond pathophysiology could improve our ability to treat symptoms. While co-morbidities such as anxiety and depression may play a role, the impact of this relationship with symptoms on our ability to perceive bodily sensations (termed interoception), or even our general and symptom-specific attention is not yet understood. Here we studied 63 individuals with asthma and 30 healthy controls. Alongside physiological tests including spirometry, bronchodilator responsiveness, expired nitric oxide and blood eosinophils, we collected self-reported questionnaires covering affective factors such as anxiety and depression, as well as asthma symptoms and asthma-related quality of life (individuals with asthma only). Participants additionally completed a breathing-related interoception task and two attention tasks designed to measure responsiveness to general temporal/spatial cues and specific asthma-related threatening words. We conducted an exploratory factor analysis across the questionnaires which gave rise to key components of Mood and Symptoms, and compared these to physiological, interoceptive and attention measures. While no relationships were found between symptoms and physiological measures in asthma alone, negative mood was related to both decreased interoceptive metacognitive sensitivity ( insight into interoceptive performance) and metacognitive bias (confidence in interoceptive decisions), as well as increased effects of spatial orienting cues in both asthma and controls. Furthermore, the relationship between the extent of symptoms and negative mood revealed potential sub-groups within asthma, with those who displayed the most severe symptoms without concurrent negative mood also demonstrating altered physiological, interoceptive and attention measures. Our findings are a step towards understanding how both symptoms and mood are related to our ability to interpret bodily symptoms, and to explore how the balance between mood and symptoms may help us understand the heterogeneity in conditions such as asthma.

neuroscience↗

bioRxiv2020

Physioxia stimulates extra-cellular matrix deposition and increases mechanical properties of human chondrocyte-derived tissue-engineered cartilage

Cartilage tissue has been recalcitrant to tissue engineering approaches. In this study, human chondrocytes were formed into self-assembled cartilage sheets, cultured in physiologic (5%) and atmospheric (20%) oxygen conditions and underwent biochemical, histological and biomechanical analysis at one- and two-months. The results indicated that sheets formed at physiological oxygen tension were thicker, contained greater amounts of glycosaminoglycans (GAGs) and type II collagen, and had greater compressive and tensile properties than those cultured in atmospheric oxygen. In all cases, cartilage sheets stained throughout for extracellular matrix components. Type II-IX-XI collagen heteropolymer formed in the neo-cartilage and fibrils were stabilized by trivalent pyridinoline cross-links. Collagen cross-links were not significantly affected by oxygen tension but increased with time in culture. Physiological oxygen tension and longer culture periods both served to increase extracellular matrix components. The foremost correlation was found between compressive stiffness and the GAG to collagen ratio. SummaryTissue-engineered cartilage formed from human articular chondrocytes produces thicker, stiffer, more extracellular-matrix rich cartilage tissue when grown under physiological (5%) vs. atmospheric oxygen (20%) tension.

cell biology↗

bioRxiv2020

Conformational changes and channel gating induced by CO2 binding to Connexin26

CO2 is the inevitable by-product of oxidative metabolism. Many physiological processes such as breathing1 and cerebral blood flow2 are sensitive to CO2. Historically, the physiological actions of CO2 have been regarded as being mediated exclusively via changes in pH. Here, we change this consensus by showing that the gap junction protein Connexin26 (Cx26) acts as a receptor for CO2 showing sensitivity to modest changes in PCO2 around the physiological norm3-6. Mass spectrometry analysis7 shows that CO2 carbamylates specific lysines on a regulatory loop of Cx26 at high, but not at low levels of PCO2. By means of high resolution cryo-EM, we have solved structures of Cx26 gap junctions at 1.9, 2.2 and 2.1 [A] for PCO2 of 90, 55 and 20 mmHg respectively, all at pH 7.4. Classification of the particles at each level of PCO2, shows the transmembrane helices and N-terminal helix flexing at the dynamic cytoplasmic side of the protein. Gating of Cx26 gap junctions by CO2 involves movements of the N-terminus to plug the channel at high PCO2. We therefore provide mechanistic detail for a new paradigm by which CO2 can directly control breathing8 and other key physiological functions9.

biochemistry↗

bioRxiv2020

Translational activity is uncoupled from nucleic acid content in bacterial cells of the human gut microbiota

BackgroundChanges in bacterial diversity in the human gut microbiome, characterized primarily though DNA sequencing methods, have been associated with many different adverse health conditions. However, these changes do not always reflect changes in bacterial activity, and thus how the gut microbiome is implicated in disease is still not often understood. New methods that link together bacterial function to bacterial identity are needed to further explore the role of the gut microbiome in health and disease. We optimized bioorthogonal non-canonical amino acid tagging (BONCAT) for the gut microbiota and combined it with fluorescently activated cell sorting and sequencing (FACS-Seq) to identify the translationally active members of the community. We then used this novel technique to compare and contrast to other methods of bulk community measurements of activity and viability: physiological staining of relative nucleic acid content and membrane damage. Relative nucleic acid content has previously been linked to metabolic activity, yet remains currently undefined for the human gut microbiota. ResultsTen healthy, unrelated individuals were sampled to determine the proportion and diversity of distinct physiological fractions of their gut microbiota. The translationally active bacteria represent about half of the gut microbiota, and are not distinct from the whole community. The high nucleic acid content (HNA) bacteria also represent about half of the gut microbiota, but are distinct from the whole community and correlate with the damaged subset. Perturbing the community with xenobiotics previously shown to alter bacterial activity but not diversity resulted in stronger changes in the distinct physiological fractions than in the whole community. ConclusionsBONCAT is a suitable method to probe the translationally active members of the human gut microbiota, and combined with FACS-Seq, allows for their identification. The high nucleic acid content bacteria are not necessarily the protein-producing bacteria in the community, and so further work is needed to understand the relationship between nucleic acid content and bacterial metabolism in the human gut. Taking into account physiologically distinct subsets of the gut microbiota may be more informative than relying on whole community profiling.

microbiology↗

bioRxiv2021

Canopy spectral reflectance detects oak wilt at the landscape scale using phylogenetic discrimination

The oak wilt disease caused by the invasive fungal pathogen Bretziella fagacearum is one of the greatest threats to oak-dominated forests across the Eastern United States. Accurate detection and monitoring over large areas are necessary for management activities to effectively mitigate and prevent the spread of oak wilt. Canopy spectral reflectance contains both phylogenetic and physiological information across the visible near-infrared (VNIR) and short-wave infrared (SWIR) ranges that can be used to identify diseased red oaks. We develop partial least square discriminant analysis (PLS-DA) models using airborne hyperspectral reflectance to detect diseased canopies and assess the importance of VNIR, SWIR, phylogeny, and physiology for oak wilt detection. We achieve high accuracy through a three-step phylogenetic process in which we first distinguish oaks from other species (90% accuracy), then red oaks from white oaks (Quercus macrocarpa) (93% accuracy), and, lastly, infected from non-infected trees (80% accuracy). Including SWIR wavelengths increased model accuracy by ca. 20% relative to models based on VIS-NIR wavelengths alone; using a phylogenetic approach also increased model accuracy by ca. 20% over a single-step classification. SWIR wavelengths include spectral information important in differentiating red oaks from other species and in distinguishing diseased red oaks from healthy red oaks. We determined the most important wavelengths to identify oak species, red oaks, and diseased red oaks. We also demonstrated that several multispectral indices associated with physiological decline can detect differences between healthy and diseased trees. The wavelengths in these indices also tended to be among the most important wavelengths for disease detection within PLS-DA models, indicating a convergence of the methods. Indices were most significant for detecting oak wilt during late August, especially those associated with canopy photosynthetic activity and water status. Our study suggests that coupling phylogenetics, physiology, and canopy spectral reflectance provides an interdisciplinary and comprehensive approach that enables detection of forest diseases at large scales. These results have potential for direct application by forest managers for detection to initiate actions to mitigate the disease and prevent pathogen spread.

ecology↗

bioRxiv2021

A Novel Swine Model of the Acute Respiratory Distress Syndrome Using Clinically-Relevant Injury Exposures

To date, existing animal models of the acute respiratory distress syndrome (ARDS) have failed to translate preclinical discoveries into effective pharmacotherapy or diagnostic biomarkers. To address this translational gap, we developed a high-fidelity swine model of ARDS utilizing clinically-relevant lung injury exposures. Fourteen male swine were anesthetized, mechanically ventilated, and surgically instrumented for hemodynamic monitoring, blood, and tissue sampling. Animals were allocated to one of three groups: 1) Indirect lung injury only: animals were inoculated by direct injection of E. coli into the kidney parenchyma, provoking systemic inflammation and distributive shock physiology; 2) Direct lung injury only: animals received volutrauma, hyperoxia, and bronchoscope-delivered gastric particles; 3) Combined indirect and direct lung injury: animals were administered both above-described indirect and direct lung injury exposures. Animals were monitored for up to 12 hours, with serial collection of physiologic data, blood samples, and radiographic imaging. Lung tissue was acquired post-mortem for pathological examination. In contrast to indirect lung injury only and direct lung injury only groups, animals in the combined indirect and direct lung injury group exhibited all of the physiological, radiographic, and histopathologic hallmarks of human ARDS: impaired gas exchange (mean PaO2/FiO2 ratio 124.8 {+/-} 63.8), diffuse bilateral opacities on chest radiographs, and extensive pathologic evidence of diffuse alveolar damage. Our novel porcine model of ARDS, built on clinically-relevant lung injury exposures, faithfully recapitulates the physiologic, radiographic, and histopathologic features of human ARDS, and fills a crucial gap in the translational study of human lung injury.

pathology↗

bioRxiv2021

Water levels affect photosynthesis and nutrient use more than salinity in a scrub Red Mangrove forest of the southeastern Florida Everglades

We investigated how mangrove-island micro-elevation (i.e., habitat: center vs. edge) affects tree physiology in a scrub mangrove forest of the southeastern Everglades. We measured leaf gas exchange rates of scrub Rhizophora mangle L. trees monthly during 2019, hypothesizing that CO2 assimilation (Anet) and stomatal conductance (gsw) would decline with increasing water levels and salinity, expecting more-considerable differences at mangrove-island edges than centers, where physiological stress is greatest. Water levels varied between 0 and 60 cm from the soil surface, rising during the wet season (May-October) relative to the dry season (November-April). Porewater salinity ranged from 15 to 30 ppt, being higher at mangrove-island edges than centers. Anet maximized at 15.1 {micro}mol m-2 s-1, and gsw was typically <0.2 mol m-2 s-1, both of which were greater in the dry than the wet season and greater at island centers than edges, with seasonal variability being roughly equal to variation between habitats. After accounting for season and habitat, water level positively affected Anet in both seasons but did not affect gsw. Our findings suggest that inundation stress (i.e., water level) is the primary driver of variation in leaf gas exchange rates of scrub mangroves in the Florida Everglades, while also constraining Anet more than gsw. The interaction between inundation stress due to permanent flooding and habitat varies with season as physiological stress is alleviated at higher-elevation mangrove-island center habitats during the dry season. Freshwater inflows during the wet season, increase water levels and inundation stress at higher-elevation mangrove-island centers, but also potentially alleviate salt and sulfide stress in soils. Thus, habitat heterogeneity leads to differences in nutrient and water acquisition and use between trees growing in island centers versus edges, creating distinct physiological controls on photosynthesis, which likely affect carbon flux dynamics of scrub mangroves in the Everglades.

plant biology↗

bioRxiv2021

The seasonal dynamics of bud dormancy in grapevine suggest a regulated checkpoint prior to acclimation

Grapevine (Vitis vinifera L.) displays wide plasticity to climate and seasonality, ranging from strongly deciduous to evergreen. Understanding the physiology of decisions to grow or quiesce is critical for improved crop management, prediction, and the adaptability of production to alternative climate scenarios. The perenniating bud (N+2) is a major economic unit and focus of study. Here we investigated the physiology and transcriptome of cv. Merlot buds grown in a temperate maritime climate from summer to spring in two consecutive years. The changes in bud respiration, hydration and internal tissue oxygen data were consistent with the transcriptome data. ABA-responsive gene processes prevailed upon the transition to a deep metabolic and cellular quiescence in the bud during autumn. Light, together with hypoxia and redox signalling presided over the resumption of nuclear and cellular growth in the transition to spring. Comparisons with transcriptome data from bud burst studies revealed a number of regulatory candidates for the orderly resumption of growth in spring, including components that may integrate light and temperature signalling. Importantly however, the bud burst forcing data, which is widely used as a measure of bud dormancy, were not consistent with the physiological and transcription data. We hypothesise the existence of a physiological checkpoint following bud set in summer, which if not met results in extreme quiescence. Collectively this is the most integrated developmental dataset of the latent bud of cultivated grapevine, and establishes a platform for systems approaches to study seasonal plasticity. One sentence summaryPhysiology and transcriptome data provide strong evidence of a regulatory checkpoint prior to acclimation and dormancy in latent grapevine buds.

plant biology↗

bioRxiv2021

A preliminary assessment of allostatic load in male mantled howler monkeys

Allostatic load refers to the wear and tear of the organism associated with the repeated activation of mechanisms that allow for homeostasis. Although allostasis (i.e., maintaining stability through change) is achieved through the orchestration of multiple systems, assessments of allostatic load in wildlife have usually relied on the measurement of single or few physiological mediators. In the present study, we aimed at constructing an allostatic load index (ALI) for male mantled howler monkeys (Alouatta palliata) based on seven physiological mediators (C-peptide, triiodothyronine, glucocorticoids, testosterone, amount of activity, foraging effort, and participation in agonistic interactions). We then examined if variation in this ALI was associated with social, ecological, and abiotic challenges. For five years, we studied ten adult males belonging to two groups. We assessed physiological mediators via behavioral observations (3,364 hours of samplings), and both urine (1,500 assayed samples) and fecal (1,500 assayed samples) sampling. We calculated an ALI based on mediator specific risk scores. Seasonality, encounters with extragroup males, mating, and centrality were positively related with male allostatic load, though only the first two had significant effects on ALI. ALI reflected the physiological and behavioral responses of males to social, ecological, and abiotic challenges. It remains for future research to determine if the observed trends correspond to normal fluctuations in mechanisms that allow male mantled howler monkeys to cope with their environment.

zoology↗

bioRxiv2021

Genetic variation in haemoglobin regulates breathing in high-altitude deer mice

Physiological systems often have emergent properties but the effects of genetic variation on physiology are often unknown, which presents a major challenge to understanding the mechanisms of phenotypic evolution. We investigated the in vivo effects on respiratory physiology of genetic variants in haemoglobin (Hb) that contribute to hypoxia adaptation in high-altitude deer mice (Peromyscus maniculatus). We created F2 inter-population hybrids of highland and lowland deer mice to test the phenotypic effects of - and {beta}-globin variants on a mixed genetic background. High-altitude genotypes were associated with breathing phenotypes that enhance O2 uptake in hypoxia, including a deeper more effective breathing pattern and an augmented hypoxic ventilatory response. These effects could not be explained by erythrocyte Hb-O2 affinity or globin gene expression in the brainstem. Therefore, adaptive variation in haemoglobin can have unexpected effects on physiology that are distinct from the canonical function of this protein in circulatory O2 transport.

evolutionary biology↗

bioRxiv2021

High and low expression of the hyperpolarization activated current (Ih) in mouse CA1 stratum oriens interneurons.

Inhibitory interneurons are among the most diverse cell types in the brain; the hippocampus itself contains more than 28 different inhibitory interneurons. Interneurons are typically classified using a combination of physiological, morphological and biochemical observations. One broad separator is action potential firing: low threshold, regular spiking vs. higher threshold, fast spiking. We found that spike frequency adaptation (SFA) was highly heterogeneous in low threshold interneurons in the mouse stratum oriens region of area CA1. Analysis with a k-means clustering algorithm parsed the data set into two distinct clusters based on a constellation of physiological parameters and reliably sorted strong and weak SFA cells into different groups. Interneurons with strong SFA fired fewer action potentials across a range of current inputs and had lower input resistance compared to cells with weak SFA. Strong SFA cells also had higher sag and rebound in response to hyperpolarizing current injections. Morphological analysis shows no difference between the two cell types and the cell types did not segregate along the dorsal-ventral axis of the hippocampus. Strong and weak SFA cells were labeled in hippocampal slices from SST:cre Ai14 mice suggesting both cells express somatostatin. Voltage-clamp recordings showed hyperpolarization activated current Ih was significantly larger in strong SFA cells compared to weak SFA cells. We suggest that the strong SFA cell represents a previously uncharacterized type of CA1 stratum oriens interneuron. Due to the combination of physiological parameters of these cells, we will refer to them as Low Threshold High Ih (LTH) cells. Key PointsO_LISpike frequency adaptation (SFA) was highly variable among stratum oriens interneurons C_LIO_LIAdapting stratum oriens interneurons were separated into two cell groups using multiple subthreshold and action potential parameters by cluster analysis C_LIO_LICells with strong SFA fired fewer action potentials for a given current injection, had lower input resistance and more sag and rebound compared to weak SFA cells C_LIO_LIThe physiological differences were not correlated with neuron morphology, location in stratum oriens, or anatomical location along the dorsal-ventral axis of the hippocampus C_LIO_LIVoltage-clamp recordings revealed that strong SFA cells had higher density of the hyperpolarization activated current Ih compared to weak SFA cells C_LI

neuroscience↗

bioRxiv2021

Infection with endosymbiotic Spiroplasma disrupts tsetse (Glossina fuscipes fuscipes) metabolic and reproductive homeostasis

Tsetse flies (Glossina spp.) house a population-dependent assortment of microorganisms that can include pathogenic African trypanosomes and maternally transmitted endosymbiotic bacteria, the latter of which mediate numerous aspects of their hosts metabolic, reproductive, and immune physiologies. One of these endosymbionts, Spiroplasma, was recently discovered to reside within multiple tissues of field captured and laboratory colonized tsetse flies grouped in the Palpalis subgenera. In various arthropods, Spiroplasma induces reproductive abnormalities and pathogen protective phenotypes. In tsetse, Spiroplasma infections also induce a protective phenotype by enhancing the flys resistance to infection with trypanosomes. However, the potential impact of Spiroplasma on tsetses viviparous reproductive physiology remains unknown. Herein we employed high-throughput RNA sequencing and laboratory-based functional assays to better characterize the association between Spiroplasma and the metabolic and reproductive physiologies of G. fuscipes fuscipes (Gff), a prominent vector of human disease. Using field-captured Gff, we discovered that Spiroplasma infection induces changes of sex-biased gene expression in reproductive tissues that may be critical for tsetses reproductive fitness. Using a Gff line composed of individuals heterogeneously infected with Spiroplasma, we observed that the bacterium and tsetse host compete for finite nutrients, which negatively impact female fecundity by increasing the length of intrauterine larval development. Additionally, we found that when males are infected with Spiroplasma, the motility of their sperm is compromised following transfer to the female spermatheca. As such, Spiroplasma infections appear to adversely impact male reproductive fitness by decreasing the competitiveness of their sperm. Finally, we determined that the bacterium is maternally transmitted to intrauterine larva at a high frequency, while paternal transmission was also noted in a small number of matings. Taken together, our findings indicate that Spiroplasma exerts a negative impact on tsetse fecundity, an outcome that could be exploited for reducing tsetse population size and thus disease transmission. Author SummaryEndosymbiotic bacteria regulate numerous aspects of their hosts reproductive physiology. Natural populations of the tsetse fly, Glossina fuscipes fuscipes (Gff), house heterogeneous infections with the bacterium Spiroplasma glossinidia. Infection with the bacterium results in the presentation of several phenotypes in both male and female Gff that would put them at a significant reproductive disadvantage when compared to their counterparts that do not house the bacterium. These Spiroplasma induced phenotypes include changes in sex-biased gene expression in the reproductive organs, a depletion in the availability of metabolically critical lipids in pregnant females that results in delayed larval development, and compromised sperm fitness. These findings indicate that Spiroplasma exerts an overall negative impact on both male and female reproductive fitness and thus likely has a profound effect on fly population structure. This outcome, in conjunction with the fact that Spiroplasma infected tsetse are unusually refractory to infection with pathogenic African trypanosomes, indicates that the bacterium could be experimentally exploited to reduce disease transmission through the fly.

microbiology↗

bioRxiv2021

Functional degeneracy in translationally active Nitrospira populations confers resilience to out-selection in partial nitritation activated sludge

Managing and engineering activated sludge wastewater treatment microbiomes for low-energy nitrogen removal requires process control strategies to stop the oxidation of ammonium at nitrite. Our ability to out-select nitrite oxidizing bacteria (NOB) from activated sludge is challenged by their metabolic and physiological diversity, warranting measurements of their in situ physiology and activity under selective growth pressures. Here, we examined the stability of nitrite oxidation in activated sludge during a press disturbance induced by treating a portion of return activated sludge with a sidestream flow containing free ammonia (FA) at 200 mg NH3-N/L. The nitrite accumulation ratio peaked at 42% by day 40 in the experimental bioreactor with the press disturbance, while it did not increase in the control bioreactor. A subsequent decrease in nitrite accumulation within the experimental bioreactor coincided with shifts in dominant Nitrospira 16S rRNA amplicon sequence variants (ASVs). We applied bioorthogonal non-canonical amino acid tagging (BONCAT) coupled with fluorescence activated cell sorting (FACS) to investigate changes in the translational activity of NOB populations throughout batch exposure to FA. BONCAT-FACS confirmed that the single Nitrospira ASV washed-out of the experimental bioreactor had reduced translational activity following exposure to FA, whereas the two Nitrospira ASVs that emerged after process acclimation were not impacted by FA. Thus, the coexistence of functionally degenerate and physiologically resistant Nitrospira populations provided resilience to the nitrite-oxidizing function during the press disturbance. These results highlight how BONCAT-FACS can resolve ecological niche differentiations within activated sludge and inform strategies to engineer and control microbiome function. ImportanceNitrogen removal from activated sludge wastewater treatment systems is an energy-intensive process due to the large aeration requirement for nitrification. This energy footprint could be minimized with engineering control strategies that wash-out nitrite oxidizing bacteria (NOB) to limit oxygen demands. However, NOB populations can have a high degree of physiological diversity, and it is currently difficult to decipher the behavior of individual taxa during applied selective pressures. Here, we utilized a new substrate analog probing approach to measure the activity of NOB at the cellular translational level in the face of an applied press disturbance to the activated sludge process. Substrate analog probing corroborated the time-series reactor sampling, showing that coexisting and functionally redundant Nitrospira provided resilience to the nitrite oxidation process. Taken together, these results highlight how substrate analog approaches can illuminate in situ ecophysiologies within shared niches, and can inform strategies to improve microbiome engineering and management.

microbiology↗

bioRxiv2021

A Hypoxia Sensitive to Hypoxia Resistant Transformation in Long-Lived Germline Mutants

Signals from the germline play a significant role in determining longevity in numerous animal models. In C. elegans, ablation of the germline leads to long life span and various other types of stress resistance. It has been reported that mutations that block oogenesis or an upstream step in germline development confer strong resistance to hypoxia. We report here that the hypoxia resistance of sterile mutants is dependent on developmental stage and age. In just a 12-hour period, sterile animals transform from hypoxia sensitive L4 larvae into highly hypoxia resistant adults. Since this transformation occurs in animals with no germline, the physiological programs that determine hypoxia sensitivity must occur independently of germline signals and instead rely on developmental signals from somatic tissues. Furthermore, we found two distinct mechanisms of hypoxia resistance in long-lived germline deficient animals. First, a DAF-16/FoxO independent mechanism that occurs in all hypoxia resistant sterile adults and, second, a DAF-16/FoxO dependent mechanism that confers an added layer of resistance, or "super-resistance", to animals with no germline as they age past day 1 of adulthood. RNAseq data showed that nearly all genes involved in both cytosolic and mitochondrial protein translation, as well as in mitochondrial protein import, are repressed in germline deficient adults and further repressed as they age. The hypoxia super-resistance of aging germline deficient animals was suppressed by dual mutation of ncl-1 and larp-1, two regulators of nucleolar biology and protein translation, demonstrating that the hypoxia super-resistance mechanism involves reduced protein translation. These studies provide novel insight into a profound physiological transformation that takes place in germline mutants during development, showing that some of the unique physiological properties of these long-lived animals are dependent on developmental repression of genes involved in protein translation, which operate independently of germline signals. AUTHOR SUMMARYIn addition to being extremely long lived, germline deficient animals have other extraordinary properties, such as robust resistance to oxygen deprivation. Here we provide new insight into the mechanisms of hypoxia resistance in germline deficient animals. We demonstrate that, in just a 12-hour period, germline mutants transform from hypoxia sensitive larvae into highly hypoxia resistant adults. Therefore, hypoxia resistance is not a general property of germline ablated animals, but is instead "switched on" only in adult animals. We have found two distinct mechanisms of hypoxia resistance in germline deficient animals and both mechanisms are mediated by signals from somatic tissues and do not require the germline. We have determined that reduced transcription of genes involved in protein translation is one of the mechanisms of hypoxia resistance. Like hypoxia resistance, repression of protein translation genes only occurs in adults. Our findings establish that the unique physiological properties of germline-deficient animals are "switched" on in adults and therefore must be mediated by developmental signals from somatic tissues. We conclude that the L4/adult developmental switch in germline ablated animals presents an excellent system for investigating the longevity and hypoxia resistance of germline deficient animals.

genetics↗

bioRxiv2021

Clinical recovery of Macaca fascicularis infected with Plasmodium knowlesi

BackgroundKra monkeys (Macaca fascicularis), a natural host of Plasmodium knowlesi, control parasitaemia caused by this parasite species and escape death without treatment. Knowledge of the disease progression and resilience in kra monkeys will aid the effective use of this species to study mechanisms of resilience to malaria. This longitudinal study aimed to define clinical, physiological and pathological changes in kra monkeys infected with P. knowlesi, which could explain their resilient phenotype. MethodsKra monkeys (n = 15, male, young adults) were infected intravenously with cryopreserved P. knowlesi sporozoites and the resulting parasitaemias were monitored daily. Complete blood counts, reticulocyte counts, blood chemistry and physiological telemetry data (n = 7) were acquired as described prior to infection to establish baseline values and then daily after inoculation for up to 50 days. Bone marrow aspirates, plasma samples, and 22 tissue samples were collected at specific time points to evaluate longitudinal clinical, physiological and pathological effects of P. knowlesi infections. ResultsAs expected, the kra monkeys controlled parasitaemia and remained with low-level, persistent parasitaemias without antimalarial intervention. Unexpectedly, early in the infection, fevers developed, which ultimately returned to baseline, as well as mild to moderate thrombocytopaenia, and moderate to severe anaemia. Mathematical modeling and the reticulocyte production index indicated that the anaemia was largely due to the removal of uninfected erythrocytes and not impaired production of erythrocytes. Mild tissue damage was observed, and tissue parasite load was associated with tissue damage even though parasite accumulation in the tissues was generally low. ConclusionsKra monkeys experimentally infected with P. knowlesi sporozoites presented with multiple clinical signs of malaria that varied in severity among individuals. Overall, the animals shared common mechanisms of resilience characterized by controlling parasitaemia 3-5 days after patency, and controlling fever, coupled with physiological and bone marrow responses to compensate for anaemia. Together, these responses likely minimized tissue damage while supporting the establishment of chronic infections, which may be important for transmission in natural endemic settings. These results provide new foundational insights into malaria pathogenesis and resilience in kra monkeys, which may improve understanding of human infections.

pathology↗

bioRxiv2021

Effect of artificial light at night on sleep and metabolism in weaver birds

Artificial light at night is constantly minimizing the span of dark nights from the natural light-dark cycle of earth. Over the past century, the lightscape of earth has completely changed owing to technological advancements which subsequently changed the lifestyle of human as well as the nearby animal species. This motivated the present study, wherein we investigated the impact of light at night (LAN) on behavior and physiology of a diurnal passerine finch, baya weaver (Ploceus philippinus). A group of bird (N=10) exposed to 12L:12D photoperiod was initially subjected to dark nights (0 lux) for a period of 10 days followed by 5 lux; night light for a span of 4 weeks. First week in LAN served as acute treatment with respect to fourth week (chronic). Acute exposure had more pronounced impact on the behavioral and physiological observations when compared with chronic treatment. The results reveal significant increase in nighttime activity, sleep loss, significant inclusion of drowsiness behavior during the day in response to LAN. Beside these behavioral alterations, changes in physiological parameters such as; reduction in body mass, loss of gradient between pre- and post-prandial blood glucose levels, elevation in plasma corticosterone levels were more prominent during acute exposure of LAN. Plasma metabolites such as triglycerides, total protein, serum glutamic-oxaloacetic transaminase (SGOT) and creatinine concentrations also hiked in response to LAN treatment. Thus, the study broadly enumerates the impact of acute and chronic exposure of LAN on behavior (rest/sleep) and physiology (metabolism) of birds.

zoology↗

bioRxiv2021

Trichoderma asperellum: First report in Bangladesh

The experiment was conducted at four laboratory, Bangladesh Agricultural University, Mymensingh to identify T. asperellum and its characterization was obtained from the rhizosphere of 49 different crops in 109 different locations of 36 districts in Bangladesh. Fifteen isolates of 88 isolates in T. asperellum were characterized on morphological and physiological features. All of the isolates were produced at 35 {degrees}C, but only TR27 and TR45 were grown and sporulate at 40 {degrees}C. Mycelial growth of all the isolates was reduced with the increasing of pH value. Molecular characterization in four isolates of TR27, TR45, TR70 and TR85 were studied at ITS and TEF region nucleotide sequencing among 15 isolates of T. asperellum and the morphological characterization at ITS and tef1 nucleotide sequencing was assimilated with molecular characterization. The isolates of TR45, TR70 and TR85 were found with 98% homology and TR27 exhibited 88% against their respective closest isolate. The isolates of TR27 and TR85 marked their respective nearest homology at 96%, while TR45 showed 99%, and 93% homology was signified with TR70 in TEF sequences. Three isolates (TR45, TR70 and TR85) were identified as T. asperellum of 100% bootstrap value and TR27 isolate was also recognized with 72% bootstrap value in phylogenetic tree. In phylogenetic analysis, tef1 extended better differentiation among the Trichoderma isolates within and among the groups of closely related species than the rDNA of the ITS region in reflecting wider variability in the isolates while a higher transversion ratio and evolutionary divergence were defined. Moreover the exploring of scientific and useful diversification of Trichoderma isolates on physiological and molecular traits has deemed in research fields. IMPORTANCETrichoderma is an important genus in different aspect of uses as biofertilizer and bioremediation in agriculture. Specific identification in physiological and molecular characterization has also foremost attention with genome sequences as stored in databases. The diversity and genetic distances were ascertained among the isolates in phylogenetic analysis with the sequence data of the ITS region and tef1 of known Trichoderma strains was found in GenBank. Consequently, the databases of Trichoderma asperellum in molecular characterization can be frequently studied in the beneficial scope of agriculture. Trichoderma isolates of Rhizosphere soil in 4 locations with crops of (TR27) Sadar, Moulvibazar (Rice), (TR45) Sadar, Mymensingh (Sweet gourd), (TR70) Chapra, Chapai Nawabganj (Sesame) and (TR85) Nayanpur, Lalmonirhat (Maize) were found to be substantiated as Trichoderma asperellum strain in Bangladesh that were confirmed on physiological and molecular characters, and utilization of this strain can run faster in invasive technologies to control crop diseases.

molecular biology↗

bioRxiv2021

Comparison of Plaque Size, Thermal Stability, and Replication Rate among SARS-CoV-2 Variants of Concern

SARS-CoV-2, like other RNA viruses, has a propensity for genetic evolution owing to the low fidelity of its viral polymerase. This evolution results in the emergence of novel variants with different characteristics than their ancestral strain. Several recent reports have described a series of novel SARS-CoV-2 variants. Some of these have been identified as variants of concern (VOCs), including alpha (B.1.1.7, Clade GRY), beta (B.1.351, Clade GH), gamma (P.1, Clade GR), and delta (B.1.617.2, Clade G). VOCs are likely to have some effect on transmissibility, antibody evasion, and changes in therapeutic or vaccine effectiveness. However, the physiological and virological understanding of these variants remains poor. We demonstrated that these four VOCs exhibited differences in plaque size, thermal stability at physiological temperature, and replication rates. The mean plaque size of beta was the largest, followed by those of gamma, delta, and alpha. Thermal stability, evaluated by measuring infectivity and half-life after prolonged incubation at physiological temperature, was correlated with plaque size in all variants except alpha. However, despite its relatively high thermal stability, alphas small plaque size resulted in lower replication rates and fewer progeny viruses. Our findings may inform further virological studies of SARS-CoV-2 variant characteristics, VOCs, and variants of interest. These studies are important for the effective management of the COVID-19 pandemic. IMPORTANCEThe global pandemic caused by SARS-CoV-2 continues to persist, due in part to mutations that have resulted in the emergence of different variants. Many of these variants have become more virulent and infectious than their ancestral strain, resulting in an ever-increasing spread. However, our virological understanding of these variants remains poor. Here, we directly compared the plaque size, stability, and replication kinetics of four SARS-CoV-2 variants of concern following prolonged incubation at physiological temperatures. Our observations may help to characterize each variant in terms of their interactions with host factors and responses to environmental conditions. We also believe that our evaluations will improve understanding of the emergence of new variants and contribute to controlling their spread.

microbiology↗