Biology subjects

Hussein, A.

Publications and source records attributed to Hussein, A..

14 recordsLinked to original sources

bioRxiv2026

Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

Multiple sclerosis (MS) is a progressive neuroinflammatory demyelinating disease of the central nervous system (CNS) that remains incurable. Autoreactive myelin-specific T cells contribute to immunopathology by directly targeting and damaging oligodendrocytes in situ. In oligodendrocytes, several immune-modulatory functions have been described that can ameliorate immune damage. However, a systematic discovery of cell-intrinsic mechanisms that protect oligodendrocytes against T cell-derived cytotoxic mechanisms has not been performed. We used human MO3.13 oligodendrocytic cells and human antigen-specific cytotoxic T cells to conduct a high-throughput (HTP) RNAi-based screen with altogether 4155 genes to identify oligodendrocyte-intrinsic immune resistance genes (IRGs). The screen revealed 133 candidate IRGs. Among them, we validated 32, which exerted a strong immuno-protective phenotype. We studied IRG expression landscapes in human brain cell subsets from postmortem brain tissues of MS and control individuals. This revealed clustered expression of IRGs in a cell-type and oligodendrocyte subset-specific manner and differential IRG expression between MS patients and controls in distinct oligodendrocyte subclusters. ChEA3 analysis revealed cell type-specific expression of transcription factors that can drive expression of respective IRGs. Explorative molecular mode of action analyses of five selected IRGs, STK11, KCNH8, ABCA2, SLC1A3 and CHRNA1 revealed that these prevented death receptor-mediated apoptosis induced by T cell-derived cytotoxic molecules. In particular, they controlled TRAIL-induced apoptosis by suppressing JNK1 activation through interfering with several upstream pathways regulating metabolic, potassium, cholesterol, glutamate and acetylcholine homeostasis. In addition, STK11, ABCA2, and CHRNA1 regulated TRAIL-R2 surface expression contributing to increased TRAIL-sensitivity whereas KCNH8 expression in oligodendrocytes inhibited secretion of inflammatory cytokines by cytotoxic T cells. Taken together, we here demonstrate the existence of multiple co-expressed IRGs in human oligodendrocytes that regulate multifaceted mechanisms of T cell resistance and are dysregulated in oligodendrocyte subsets of MS patients.

immunology↗

bioRxiv2026

Lipids are essential for potassium transport by KdpFABC from E. coli

KdpFABC is a hetero-tetrameric potassium pump that uses ATP to import potassium and thereby maintain homeostasis in bacteria under stress conditions. KdpA is a channel-like subunit with a selectivity filter that binds potassium from the periplasm. K+ then moves through a [~]40[A]-long intramembrane tunnel to reach a canonical binding site in KdpB. KdpB is a P-type ATPase that orchestrates conformational changes associated with the Post-Albers reaction cycle, involving E1 and E2 conformations and formation of an aspartyl phosphate intermediate as a way of coupling ATP hydrolysis to K+ transport. To elucidate the associated structural changes in a lipid environment, we reconstituted wild-type KdpFABC into lipid nanodiscs and used cryo-EM to image the complex under active turnover. The resulting six high resolution (2.1-2.7 [A]) structures provide new insight into the sequence of allosteric changes that produce (1) occlusion of K+ at the canonical binding site and (2) expulsion of K+ from this site and into a low-affinity release site. The structures also reveal two types of lipids bound to the complex. Specifically, two structural lipids bind at subunit interfaces and [~]20 annular lipids are seen at the periphery of the complex. In addition, we tested functional effects of mutations to residues at the KdpA/KdpB interface. ATPase and transport assays were used to document functional defects that reflect delipidation of structurally compromised complexes. We conclude that lipids play an integral role in structure and function of the KdpFABC complex. SignificanceKdpFABC uses ATP to transport potassium across the plasma membrane of E. coli. To further our understanding of its mechanism, we put purified KdpFABC molecules into membrane bilayers and used cryo-EM to capture structures during active transport. We have thereby produced structures representing all major states of the transport cycle with a high degree of precision. Analysis of these structures reveals new details about two key steps in this cycle and shows lipid molecules bound to the protein. We then introduced mutations at the interface between the two main subunits, which controls passage of potassium across the membrane. Activity measurements reveal how the protein depends on lipid to stabilize the structure and facilitate transport.

biophysics↗

bioRxiv2025

Conduction pathway for potassium through the E. coli pump KdpFABC

Under osmotic stress, bacteria express a heterotetrameric protein complex, KdpFABC, which functions as an ATP-dependent K+ pump to maintain intracellular potassium levels. The subunit KdpA belongs to the Superfamily of K+ Transporters and adopts a pseudo-tetrameric architecture with a membrane embedded selectivity filter as seen in K+ channels. KdpB belongs to the superfamily of P-type ATPases with a conserved binding site for ions within the membrane domain and three cytoplasmic domains that orchestrate ATP hydrolysis via an aspartyl phosphate intermediate. Previous work has hypothesized that K+ moves parallel to the membrane plane through a 40-[A] long tunnel that connects the selectivity filter of KdpA with a canonical binding site in KdpB. In the current work, we have reconstituted KdpFABC into lipid nanodiscs and used cryo-EM to image the wild-type pump under turnover conditions. We present a 2.1 [A] structure of the E1[~]P{middle dot}ADP conformation, which reveals new features of the conduction pathway. This map shows strong densities within the selectivity filter and at the canonical binding site, consistent with K+ bound at each of these sites in this conformation. Many water molecules occupy a vestibule and the proximal end of the tunnel, which becomes markedly hydrophobic and dewetted at the subunit interface. We go on to use ATPase and ion transport assays to assess effects of numerous mutations along this proposed conduction pathway. The results confirm that K+ ions pass through the tunnel and support the existence of a low affinity site in KdpB for releasing these ions to the cytoplasm. Taken together, these data shed new light on the unique partnership between a transmembrane channel and an ATP-driven pump in maintaining the large electrochemical K+ gradient essential for bacterial survival.

biophysics↗

bioRxiv2025

Foxi2 and Sox3 are master regulators controlling ectoderm germ layer specification

In vertebrates, germ layer specification represents a critical transition where pluripotent cells acquire lineage-specific identities. We identify the maternal transcription factors Foxi2 and Sox3 to be pivotal master regulators of ectodermal germ layer specification in Xenopus. Ectopic co-expression of Foxi2 and Sox3 in prospective endodermal tissue induces the expression of ectodermal markers while suppressing mesendodermal markers. Transcriptomics analyses reveal that Foxi2 and Sox3 jointly and independently regulate hundreds of ectodermal target genes. During early cleavage stages, Foxi2 and Sox3 pre-bind to key cis-regulatory modules (CRMs), marking sites that later recruit Ep300 and facilitate H3K27ac deposition, thereby shaping the epigenetic landscape of the ectodermal genome. These CRMs are highly enriched within ectoderm-specific super-enhancers (SEs). Our findings highlight the pivotal role of ectodermal SE-associated CRMs in precise and robust ectodermal gene activation, establishing Foxi2 and Sox3 as central architects of ectodermal lineage specification. HighlightsFoxi2 and Sox3 are master regulators for the ectodermal germ layer and sub-lineages Five ectodermal cell states of early gastrulae are regulated by Foxi2 and Sox3 Foxi2 and Sox3 binding sites in the genome are enriched in ectoderm super-enhancers SE-associated genes show high expression levels, low noise, stabilizing ectodermal regulation

developmental biology↗

bioRxiv2023

Development of prefrontal corticostriatal connectivity in mice

Rational decision making is grounded in learning to associate actions with outcomes, a process that depends on projections from prefrontal cortex to dorsomedial striatum. Symptoms associated with a variety of human pathological conditions ranging from schizophrenia and autism to Huntingtons and Parkinsons disease point toward functional deficits in this projection, but its development is not well understood, making it difficult to investigate how perturbations in development of this circuitry could contribute to pathophysiology. We applied a novel strategy based on Hotspot Analysis to assess the developmental progression of anatomical positioning of prefrontal cortex to striatal projections. Corticostriatal axonal territories established at P7 expand in concert with striatal growth but remain largely unchanged in positioning through adulthood, indicating they are generated by directed, targeted growth and not modified extensively by postnatal experience. Consistent with these findings, corticostriatal synaptogenesis increased steadily from P7 to P56, with no evidence for widescale pruning. As corticostriatal synapse density increased over late postnatal ages, the strength of evoked PFC input onto dorsomedial striatal projection neurons also increased, but spontaneous glutamatergic synaptic activity was stable. Based on its pattern of expression, we asked whether the adhesion protein, Cdh8, influenced this progression. In mice lacking Cdh8 in PFC corticostriatal projection neurons, axon terminal fields in dorsal striatum shifted ventrally. Corticostriatal synaptogenesis was unimpeded, but spontaneous EPSC frequency declined and mice failed to learn to associate an action with an outcome. Collectively these findings show that corticostriatal axons grow to their target zone and are restrained from an early age, do not undergo postnatal synapse pruning as the most dominant models predict, and that a relatively modest shift in terminal arbor positioning and synapse function has an outsized, negative impact on corticostriatal-dependent behavior.

neuroscience↗

bioRxiv2023

Energy Coupling and Stoichiometry of Zn2+/H+ Antiport by the Cation Diffusion Facilitator YiiP

YiiP is a prokaryotic Zn2+/H+ antiporter that serves as a model for the Cation Diffusion Facilitator (CDF) superfamily, members of which are generally responsible for homeostasis of transition metal ions. Previous studies of YiiP as well as related CDF transporters have established a homodimeric architecture and the presence of three distinct Zn2+ binding sites named A, B, and C. In this study, we use cryo-EM, microscale thermophoresis and molecular dynamics simulations to address the structural and functional roles of individual sites as well as the interplay between Zn2+ binding and protonation. Structural studies indicate that site C in the cytoplasmic domain is primarily responsible for stabilizing the dimer and that site B at the cytoplasmic membrane surface controls the structural transition from an inward facing conformation to an occluded conformation. Binding data show that intramembrane site A, which is directly responsible for transport, has a dramatic pH dependence consistent with coupling to the proton motive force. A comprehensive thermodynamic model encompassing Zn2+ binding and protonation states of individual residues indicates a transport stoichiometry of 1 Zn2+ to 2-3 H+ depending on the external pH. This stoichiometry would be favorable in a physiological context, allowing the cell to use the proton gradient as well as the membrane potential to drive the export of Zn2+.

biophysics↗

bioRxiv2022

Cognitive Deficits and Altered Cholinergic Innervation in Young Adult Mice Carrying a Parkinson's Disease LRRK2-G2019S Knockin Mutation

Impaired executive function is a common and debilitating non-motor symptom of idiopathic and hereditary Parkinsons disease (PD), but there is little understanding of the underlying pathophysiological mechanisms and circuits. The G2019S mutation in the kinase domain of leucine-rich repeat kinase 2 (LRRK2) greatly increases risk for late-onset PD, and non-manifesting LRRK2-G2019S carriers also exhibit early and significant cognitive impairment. Here, we subjected young adult mice carrying a Lrrk2-G2019S knockin mutation to touchscreen-based operant tasks that measure attention, goal-directed learning and cognitive flexibility, all of which rely on prefrontal-striatal connectivity and are strongly modulated by cholinergic innervation. In a visuospatial attention task, mutant mice exhibited significantly more omissions and longer response latencies than controls that could not be attributed to deficits in motivation, visual sensory perception per se or locomotion, thereby suggesting impairment in divided attention and slower information processing speed. Pretreating mice with the acetylcholinesterase inhibitor donepezil normalized both higher omission rates and longer reward latencies in the mutants, but did not affect any performance metric in controls. Strikingly, cholinergic fiber density in mPFC and dorsomedial striatum was significantly sparser in mutants than in controls, while further behavioral interrogation of the mutants revealed significant impairments in action-outcome associations but preserved cognitive flexibility. These data suggest that the G2019S mutation impacts cholinergic innervation and impairs corticostriatal network function in young adulthood that may contribute to early PD-associated cognitive deficits. STATEMENT OF SIGNIFICANCEThe LRRK2-G2019S mutation causes hereditary Parkinsons disease and is found in some idiopathic cases. Early cognitive impairment is a common symptom of hereditary and idiopathic PD, yet there is little mechanistic understanding of such impairment. Here, we tested young adult Lrrk2-G2019S knockin mice in a series of touchscreen-based visuospatial tasks. We found that mutants exhibited significant deficits in attention and goal-directed learning, and had significantly slower information processing speed. Treatment with an acetylcholinesterase inhibitor reversed some of these behavioral deficits, while anatomical analyses showed significantly sparser cholinergic innervation of brain structures important for executive function. These findings suggest the G2019S mutation alters cholinergic signaling in young adulthood, and thus may contribute to early PD-associated impairment in several cognitive domains.

neuroscience↗

bioRxiv2021

COMPARISON OF WOODY SPECIES DIVERSITY AND POPULATION STRUCTURE STATUS ACROSS DIFFERENT MANAGEMENT PRACTICE IN THE GOLA NATURAL VEGETATION, EASTER HARARGHE, OROMIA OF ETHIOPIA

The research was carried out at Gola natural vegetation eastern Ethiopia, to identify and Woody species' diversity, richness, evenness, and population structural status will be documented, as well as their diversity, richness, evenness, and population structural status will be analyzed. Using systematic sampling procedures, the woody species diversity and population structure of species were examined in 73 quadrats, each measuring 20 m x 20 m for trees and 5 m x 5 m for shrubs and climbers, within three land use systems: farm land (FL), grazing land (GL), and protected area (PA). The diameter at breast height (DBH), richness, evenness, and density of woody species were all measured in the vegetation. The Shannon Weiner Diversity Index was used to examine the diversity of vegetation. A total of 52 woody species belonging to 33 genera, and there were 24 families found. in Gola natural vegetation. Fabaceae was represented by the highest number of species comprising 8 (18.18 %), 9 (25.00 %) and 8 (32%) of the total number of plant species found in PA, GL, and FL. The PA site had significantly highest population density of vegetation, followed by the GL site, while the FL site had the lowest. The total basal area of PA, GL and FL were 43.73, 31.68 and 22.62 m2/ha, respectively. PA site had significantly (P= 0.042) highest Shannons diversity index value with mean (3.53) than the others two land use system. This result suggests significance of anthropological disturbance like grazing and farming on woody species diversity and natural forest ecosystem appeared to be adverse dependent on the category and severity of the anthropogenic disturbances.

ecology↗

bioRxiv2021

Tributyltin Protects Against Ovariectomy-Induced Trabecular Bone Loss in C57BL/6J Mice with an Attenuated Effect in High Fat Fed Mice

Risk factors for poor bone quality include estrogen loss at menopause, a high fat diet and exposures to drugs/chemicals that activate peroxisome proliferator activated receptor gamma (PPAR{gamma}). We observed that the PPAR{gamma} and retinoid X receptor dual ligand, tributyltin (TBT), repressed periosteal bone formation but enhanced trabecular bone formation in female C57BL6/J mice. Here, we examined the interaction of diet, ovariectomy (OVX) and TBT exposure on bone structure. C57BL/6J mice underwent either sham surgery or OVX at 10 weeks of age. At 12 weeks of age, they were placed on a low (10% kcal) or high (45% kcal) fat, sucrose-matched diet and treated with Vh or TBT (1 or 5 mg/kg) for 14 weeks. OVX increased body weight gain in mice on either diet. TBT enhanced body weight gain in intact mice fed a high fat diet, but decreased weight gain in OVX mice. Elemental tin concentrations increased dose-dependently in bone. TBT had marginal effects on cortical and trabecular bone in intact mice fed a low- or high- fat diet. OVX caused a reduction in cortical and trabecular bone, regardless of diet. In high-fat fed OVX mice, TBT further reduced cortical thickness, bone area and total area. Interestingly, TBT protected against OVX-induced trabecular bone loss in low fat fed mice. The protective effect of TBT was nullified by the high fat diet and accompanied by a significant decrease in serum bone formation markers. Our novel observations will provide new information on basic bone biology, potential therapeutic targets and toxicological pathways.

pharmacology and toxicology↗

bioRxiv2021

Structure and dynamics of a cold-active esterase reveals water entropy and active site accessibility as the likely drivers for cold-adaptation

Cold-active esterases hold great potential for undertaking useful biotransformations at low temperatures. Here, we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical /{beta} hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7s closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Comparison of B-factors with the closest related mesophilic and thermophilic esterases suggests the EstN7 cap region is proportionally less flexible. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are the main drivers of EstN7s cold adaptation rather than changes in dynamics.

biochemistry↗

bioRxiv2021

Structure guided engineering of a cold active esterase expands substrate range though a stabilisation mutation that allows access to a buried water chamber

Cold active esterases represent an important class of enzymes capable of undertaking useful chemical transformations at low temperatures. EstN7 from Bacillus cohnii represents a true psychrophilic esterase with a temperature optimum below 20{degrees}C. We have recently determined the structure of EstN7 and have used this knowledge to understand substrate specificity and expands its substrate range through protein engineering. Substrate range is determined by a plug at the end of acyl binding pocket that blocks access to a buried water filled cavity, so limiting EstN7 to turnover of C2 and C4 substrates. Data mining revealed a potentially important commercial reaction, conversion of triacetin to only the 1,2-glyceryl diacetate isomer, which the EstN7 could achieve. Residues M187, N211 and W206 were identified as plug residues. M187 was identified as the key plug residue but mutation to alanine destabilised the structure as whole. Another plug mutation, N211A had a stabilising effect on EstN7 and suppressed the destabilising M187A mutation. The M187A-N211A variant had the broadest substrate range, capable of hydrolysing a C8 substrate. Thus, the structure of EstN7 together with focused engineering has provided new insights into the structural stability and substrate specificity that allowed expansion of substrate range.

biochemistry↗

bioRxiv2021

SARS-CoV-2 infection reduces Krüppel-Like Factor 2 in human lung autopsy

Acute respiratory distress syndrome (ARDS) occurred in ~12% of hospitalized COVID-19 patients in a recent New York City cohort. Pulmonary endothelial dysfunction, characterized by increased expression of inflammatory genes and increased monolayer permeability, is a major component of ARDS. Vascular leak results in parenchymal accumulation of leukocytes, protein, and extravascular water, leading to pulmonary edema, ischemia, and activation of coagulation associated with COVID-19. Endothelial inflammation further contributes to uncontrolled cytokine storm in ARDS. We have recently demonstrated that Kruppel-like factor 2 (KLF2), a transcription factor which promotes endothelial quiescence and monolayer integrity, is significantly reduced in experimental models of ARDS. Lung inflammation and high-tidal volume ventilation result in reduced KLF2, leading to pulmonary endothelial dysfunction and acute lung injury. Mechanistically, we found that KLF2 is a potent transcriptional activator of Rap guanine nucleotide exchange factor 3 (RAPGEF3) which orchestrates and maintains vascular integrity. Moreover, KLF2 regulates multiple genome-wide association study (GWAS)-implicated ARDS genes. Whether lung KLF2 is regulated by SARS-CoV-2 infection is unknown. Here we report that endothelial KLF2 is significantly reduced in human lung autopsies from COVID-19 patients, which supports that ARDS due to SARS-CoV-2 is a vascular phenotype possibly attributed to KLF2 down-regulation. We provide additional data demonstrating that KLF2 is down-regulated in SARS-CoV infection in mice.

pathology↗

bioRxiv2019

The Arabian camel, Camelus dromedarius interferon epsilon: functional expression, in vitro refolding, purification and cytotoxicity on breast cancer cell lines MDA-MB-231 and MCF-7

The current study highlights for the first time cloning, overexpression, purification, and assessing the cytotxcity of the novel interferon epsilon (IFN{varepsilon}), from the Arabian camel Camelus dromedarius, against two human breast cancer cell lines MDA-MB-231 and MCF-7. Full-length cDNA encoding interferon epsilon (IFN{varepsilon}) was isolated and cloned from the liver of the Arabian camel, C. dromedarius using reverse transcription-polymerase chain reaction. The sequence analysis of the camel IFN{varepsilon} cDNA showed a 582-bp open reading frame encoding a protein of 193 amino acids with an estimated molecular weight of 22.953 kDa. A BLAST search analysis revealed that the C. dromedarius IFN{varepsilon} shared high sequence identity with the IFN genes of other species, such as Camelus ferus, Vicugna pacos, and Homo sapiens. Expression of the camel IFN{varepsilon} cDNA in Escherichia coli gave a fusion protein band of 22.73 kDa after induction with either isopropyl {beta}-D-1-thiogalactopyranoside or lactose for 5 h. Recombinant IFN{varepsilon} protein was overexpressed in the form of inclusion bodies that were easily solubilized and refolded using SDS and KCl. The solubilized inclusion bodies were purified to apparent homogeneity using nickel affinity chromatography. We examined the effect of IFN{varepsilon} on two breast cancer cell lines MDA-MB-231 and MCF-7. In both cell lines, IFN{varepsilon} inhibited cell survival in a dose dependent manner as observed by MTT assay, morphological changes and apoptosis assay. Caspase-3 expression level was found to be increased in MDA-MB-231 treated cells as compared to untreated cells.

molecular biology↗

bioRxiv2019

The COMPARE Data Hubs

Data sharing enables research communities to exchange findings and build upon the knowledge that arises from their discoveries. Areas of public and animal health as well as food safety would benefit from rapid data sharing when it comes to emergencies. However, ethical, regulatory, and institutional challenges, as well as lack of suitable platforms which provide an infrastructure for data sharing in structured formats often lead to data not being shared, or at most shared in form of supplementary materials in journal publications. Here, we describe an informatics platform that includes workflows for structured data storage, managing and pre-publication sharing of pathogen sequencing data and its analysis interpretations with relevant stakeholders.

bioinformatics↗