Biology subjects

Martinez, D.

Publications and source records attributed to Martinez, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Fluorescence fluctuation based super resolution microscopy, basic concepts for an easy start.

Due to the wave nature of light, optical microscopy has a lower-bound lateral resolution limit of about half of the wavelength of the detected light, i.e., within the range of 200 to 300 nm. The Fluorescence Fluctuation based Super Resolution Microscopy (FF-SRM) encompases a collection of image analysis techniques which rely on the statistical processing of temporal variations of fluorescence to reduce the uncertainty about the fluorophore positions within a sample, hence, bringing spatial resolution down to several tens of nm. The FF-SRM is known to be suitable for live-cell imaging due to its compatibility with most fluorescent probes and lower instrumental and experimental requirements, which are mostly camera-based epifluorescence instruments. Each FF-SRM approach has strengths and weaknesses, which depend directly on the underlying statistical principles through which enhanced spatial resolution is achieved. In this review, the basic concepts and principles behind a range of FF-SRM methods published to date are revisited. Their operational parameters are explained and guidance for its selection is provided.

bioinformatics↗

bioRxiv2022

Vertical HIV-1 transmission in the setting of maternal broad and potent antibody responses

Despite the worldwide availability of antiretroviral therapy (ART), approximately 150,000 pediatric HIV infections continue to occur annually. ART can dramatically reduce HIV mother-to-child transmission (MTCT), but inconsistent drug access and adherence, as well as primary maternal HIV infection during pregnancy and lactation are major barriers to eliminating vertical HIV transmission. Thus, immunologic strategies to prevent MTCT, such as an HIV vaccine, will be required to attain an HIV-free generation. A primary goal of HIV vaccine research has been to elicit broadly neutralizing antibodies (bnAbs) given the ability of passive bnAb immunization to protect against sensitive strains, yet we previously observed that HIV-transmitting mothers have more plasma neutralization breadth than non-transmitting mothers. Additionally, we have identified infant transmitted/founder (T/F) viruses that escape maternal bnAb responses. In this study, we examine a cohort of postpartum HIV-transmitting women with neutralization breadth to determine if certain maternal bnAb specificities drive the selection of infant T/F viruses. Using HIV pseudoviruses that are resistant to neutralizing antibodies targeting common bnAb epitopes, we mapped the plasma bnAb specificities of this cohort. Significantly more transmitting women with plasma bnAb activity had a mappable plasma bnAb specificity (six of seven, or 85.7%), compared to that of non-transmitting women with plasma bnAb activity (seven of twenty-one, or 33.3%, p=0.029 by 2-sided Fisher exact test). Our study suggests that having multispecific broad activity and/or uncommon epitope-specific bnAbs in plasma may be associated with protection against the vertical HIV transmission in the setting of maternal bnAb responses. ImportanceAs mother to child transmission (MTCT) of HIV plays a major part in the persistence of the HIV/AIDS epidemic and bnAb-based passive and active vaccines are a primary strategy for HIV prevention, research in this field is of great importance. While previous MTCT research has investigated the neutralizing antibody activity of HIV-infected women, this is to our knowledge the largest study identifying differences in bnAb specificity of maternal plasma between transmitting and non-transmitting women. Here, we show that among HIV-infected women with broad and potent neutralization activity, more postpartum-transmitting women had a mappable plasma broadly neutralizing antibody (bnAb) specificity, compared to that of non-transmitting women, suggesting that the non-transmitting women more often have multi-specific bnAb responses or bnAb responses that target uncommon epitopes. Such responses may be required for protection against vertical HIV transmission in the setting of maternal bnAb responses.

immunology↗

bioRxiv2022

Inter-animal variability in activity phase is constrained by synaptic dynamics in an oscillatory network

The levels of voltage-gated and synaptic currents in the same neuron type can vary substantially across individuals. Yet, the phase relationships between neurons in oscillatory circuits are often maintained, even in the face of varying oscillation frequencies. We examined whether synaptic and intrinsic currents are matched to maintain constant activity phases across preparations, using the lateral pyloric (LP) neuron of the stomatogastric ganglion of the crab, Cancer borealis. LP produces stable oscillatory bursts upon release from inhibition, with an onset phase that is independent of oscillation frequency. We quantified the parameters that define the shape of the synaptic current inputs across preparations and found no linear correlations with voltage-gated currents. However, several synaptic parameters were correlated with oscillation period and burst onset phase, suggesting they may play a role in phase maintenance. We used the dynamic clamp to apply artificial synaptic inputs and found that those synaptic parameters correlated with phase and period were ineffective in influencing burst onset. Instead, parameters that showed the least variability across preparations had the greatest influence. Thus, parameters that influence circuit phasing are constrained across individuals, while those that have little effect simply co-vary with phase and frequency.

neuroscience↗

bioRxiv2021

The Role of Mentoring in Promoting Diversity, Equity, and Inclusion in STEM Education and Research

Mentoring success is derived from active and respectful listening and the willingness to learn and accept opportunities for personal growth. Mentoring shapes every trainee and their career path in science, technology, engineering, and mathematics (STEM). Productive mentoring relationships cultivate rapport, stimulate moments of introspection, and provide constructive feedback. Effective mentoring in STEM allows trainees, especially underrepresented minorities (URMs), to flourish in welcoming and supportive environments. However, URM trainees often experience inadequate mentoring due to their mentors inexperience with URM groups, poor mentorship training, or a lack of understanding of their mentees journey. To promote diversity, equity, and inclusion in STEM education and research, it is essential for mentors and mentees to work together with creativity, authenticity, and networking. In this workshop, we will focus on mentees perspective on how mentors can enhance their training, professional and career development, and improve their focus. We analyzed data on feedback obtained from students interested in pursuing graduate education who attended a recent workshop. Our results show that despite low initial expectations for the workshop, many students were satisfied with the knowledge they learned. The future of increasing the URM representation in STEM lies in providing adequate community support and mentorship throughout the careers of URM professionals.

scientific communication and education↗

bioRxiv2021

An effective workshop on How to be an Effective Mentor for Underrepresented STEM Trainees

Despite an increase in programming to promote persons excluded by their ethnicity or race (PEER) scholars, minorities remain underrepresented in many STEM programs. The academic pipeline is largely leaky for underrepresented minority (URM) scholars due to a lack of effective mentorship. Many URM students experience microaggressions and discrimination from their mentors due to a lack of quality mentorship training. In this workshop, we provide a framework for how to be an effective mentor to URM trainees. Mentees, especially URM trainees, can flourish in effective mentoring environments where they feel welcomed and can comfortably develop new ideas without feeling threatened by external factors. Effective mentoring environments provide motivational support, empathy, cultural competency, and training.

scientific communication and education↗

bioRxiv2021

The Importance of Mentors and How to Handle More Than One Mentor

IntroductionWorking with multiple mentors is a critical way for students to expand their network, gain opportunities, and better prepare for future scholastic or professional ventures. However, students from underrepresented groups (UR) are less likely to be mentored or have access to mentors, particularly in science, technology, engineering, and mathematics (STEM) fields. We developed and implemented a workshop, to provide the necessary foundation for students to be better prepared for establishing future mentorships throughout graduate and professional school. MethodsFaculty well-versed in the area of effective mentorship from multiple universities developed and delivered a 1.5-hour workshop to address the roles of a mentor, especially when it comes to UR students, and how students may effectively work with multiple mentors. This workshop was delivered to a group of students from the HBCU Winston Salem State University, and a pre/post-Likert scale-based survey was administered. ResultsWe analyzed the raw data with nonparametric tests for comparison within paired samples. Wilcoxon matched-pairs and signed-rank tests showed statistically significant growth in student self-ratings related to the workshop learning objectives. ConclusionsThe "How to Handle More than One Mentor to Achieve Excellence" workshop was well received as a component of pre-graduate and pre-professional training. Incorporating workshops like this may increase student preparedness around developing and cultivating healthy mentorship relationships throughout STEM training. EDUCATIONAL OBJECTIVESBy the end of this workshop, learners will be able to: O_LIDescribe the role of mentors in developing the next generation of trainees. C_LIO_LIDescribe current research on mentorship among underrepresented populations. C_LIO_LIApply skills on effective communication needed in the development of successful mentorship relationships. C_LIO_LIWork with multiple mentors at one time while maintaining solid professional and personal relationships. C_LI

scientific communication and education↗

bioRxiv2021

Using a Champion-Oriented Mindset to Overcome the Challenges of Graduate School

Despite efforts to increase diversity, a glaring underrepresentation of minorities (URM) persists in the fields of science, technology, engineering, and mathematics (STEM). Graduate school can be a stressful step in the STEM pipeline, especially for students previously unaware of the structure and challenges of post-graduate education. To promote successful minority participation in STEM and prepare prospective students for the impending challenges of graduate school, we developed a workshop based on the mentoring and fostering of a champion-oriented mindset entitled, "The Trials and Tribulations of Graduate School: How Do You Make an Impact?". We administered the workshop to a cohort of university undergraduates and conducted pre- and post-workshop surveys to measure students perceived need for instruction on specific workshop topics. The results suggest that the workshop was well received by the students and provided information that they considered helpful to help navigate the graduate school process.

scientific communication and education↗

bioRxiv2021

Nanoscopic resolution within a single imaging frame

Mean-Shift Super Resolution (MSSR) is a principle based on the Mean Shift theory that extends spatial resolution in fluorescence images, beyond the diffraction limit. MSSR works on low- and high-density fluorophore images, is not limited by the architecture of the detector (EM-CCD, sCMOS, or photomultiplier-based laser scanning systems) and is applicable to single images as well as temporal series. The theoretical limit of spatial resolution, based on optimized real-world imaging conditions and analysis of temporal image series, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other analytical super resolution image approaches. Altogether, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications.

bioinformatics↗

bioRxiv2021

Development of GPC2-directed chimeric antigen receptors using mRNA for pediatric brain tumors

Pediatric brain tumors are the leading cause of cancer death in children with an urgent need for innovative therapies. Here we show that the cell surface oncoprotein glypican 2 (GPC2) is highly expressed on multiple lethal pediatric brain tumors, including medulloblastomas, embryonal tumors with multi-layered rosettes, other CNS embryonal tumors, as well as definable subsets of highly malignant gliomas. To target GPC2 on these pediatric brain tumors with adoptive cellular therapies and mitigate potential inflammatory neurotoxicity, we developed four mRNA chimeric antigen receptor (CAR) T cell constructs using the highly GPC2-specific fully human D3 single chain variable fragment. First, we validated and prioritized these CARs using in vitro cytotoxicity and T cell degranulation assays with GPC2-expressing neuroblastoma cells. Next, we expanded the testing of the two most potent GPC2-directed CAR constructs prioritized from these studies to GPC2-expressing medulloblastoma and high-grade glioma cell lines, showing significant GPC2-specific cell death in multiple models. Finally, locoregional delivery bi-weekly of two to four million GPC2-directed mRNA CAR T cells induced significant and sustained tumor regression in two orthotopic medulloblastoma models, and significantly prolonged survival in an aggressive orthotopic thalamic diffuse midline glioma model. No GPC2-directed CAR T cell related neurologic or systemic toxicity was observed. Taken together, these data show that GPC2 is a highly differentially expressed cell surface protein on multiple malignant pediatric brain tumors that can be targeted safely with local delivery of mRNA CAR T cells. One Sentence SummaryGlypican 2 is expressed on the surface of multiple pediatric brain tumors and can be successfully targeted with mRNA chimeric antigen receptor T cells.

cancer biology↗

bioRxiv2021

Protective efficacy of rhesus adenovirus COVID-19 vaccines against mouse-adapted SARS-CoV-2

The global COVID-19 pandemic has sparked intense interest in the rapid development of vaccines as well as animal models to evaluate vaccine candidates and to define immune correlates of protection. We recently reported a mouse-adapted SARS-CoV-2 virus strain (MA10) with the potential to infect wild-type laboratory mice, driving high levels of viral replication in respiratory tract tissues as well as severe clinical and respiratory symptoms, aspects of COVID-19 disease in humans that are important to capture in model systems. We evaluated the immunogenicity and protective efficacy of novel rhesus adenovirus serotype 52 (RhAd52) vaccines against MA10 challenge in mice. Baseline seroprevalence is lower for rhesus adenovirus vectors than for human or chimpanzee adenovirus vectors, making these vectors attractive candidates for vaccine development. We observed that RhAd52 vaccines elicited robust binding and neutralizing antibody titers, which inversely correlated with viral replication after challenge. These data support the development of RhAd52 vaccines and the use of the MA10 challenge virus to screen novel vaccine candidates and to study the immunologic mechanisms that underscore protection from SARS-CoV-2 challenge in wild-type mice. ImportanceWe have developed a series of SARS-CoV-2 vaccines using rhesus adenovirus serotype 52 (RhAd52) vectors, which exhibits a lower seroprevalence than human and chimpanzee vectors, supporting their development as novel vaccine vectors or as an alternative Ad vector for boosting. We sought to test these vaccines using a recently reported mouse-adapted SARS-CoV-2 (MA10) virus to i) evaluate the protective efficacy of RhAd52 vaccines and ii) further characterize this mouse-adapted challenge model and probe immune correlates of protection. We demonstrate RhAd52 vaccines elicit robust SARS-CoV-2-specific antibody responses and protect against clinical disease and viral replication in the lungs. Further, binding and neutralizing antibody titers correlated with protective efficacy. These data validate the MA10 mouse model as a useful tool to screen and study novel vaccine candidates, as well as the development of RhAd52 vaccines for COVID-19.

microbiology↗

bioRxiv2021

A broadly neutralizing antibody protects against SARS-CoV, pre-emergent bat CoVs, and SARS-CoV-2 variants in mice

SARS-CoV in 2003, SARS-CoV-2 in 2019, and SARS-CoV-2 variants of concern (VOC) can cause deadly infections, underlining the importance of developing broadly effective countermeasures against Group 2B Sarbecoviruses, which could be key in the rapid prevention and mitigation of future zoonotic events. Here, we demonstrate the neutralization of SARS-CoV, bat CoVs WIV-1 and RsSHC014, and SARS-CoV-2 variants D614G, B.1.1.7, B.1.429, B1.351 by a receptor-binding domain (RBD)-specific antibody DH1047. Prophylactic and therapeutic treatment with DH1047 demonstrated protection against SARS-CoV, WIV-1, RsSHC014, and SARS-CoV-2 B1.351infection in mice. Binding and structural analysis showed high affinity binding of DH1047 to an epitope that is highly conserved among Sarbecoviruses. We conclude that DH1047 is a broadly neutralizing and protective antibody that can prevent infection and mitigate outbreaks caused by SARS-like strains and SARS-CoV-2 variants. Our results argue that the RBD conserved epitope bound by DH1047 is a rational target for pan Group 2B coronavirus vaccines.

microbiology↗

bioRxiv2021

Early therapy with remdesivir and antibody combinations improves COVID-19 disease in mice

Improving the standard of clinical care for individuals infected with SARS-CoV-2 variants is a global health priority. Small molecule antivirals like remdesivir (RDV) and biologics such as human monoclonal antibodies (mAb) have demonstrated therapeutic efficacy against SARS-CoV-2, the causative agent of COVID-19. However, it is not known if combination RDV/mAb will improve outcomes over single agent therapies or whether antibody therapies will remain efficacious against variants. In kinetic studies in a mouse-adapted model of ancestral SARS-CoV-2 pathogenesis, we show that a combination of two mAbs in clinical trials, C144 and C135, have potent antiviral effects against even when initiated 48 hours after infection. The same antibody combination was also effective in prevention and therapy against the B.1.351 variant of concern (VOC). Combining RDV and antibodies provided a modest improvement in outcomes compared to single agents. These data support the continued use of RDV to treat SARS-CoV-2 infections and support the continued clinical development of the C144 and C135 antibody combination to treat patients infected with SARS-CoV-2 variants.

microbiology↗

bioRxiv2020

Possible biased virulence attenuation in the Senegal strain of Ehrlichia ruminantium by ntrX gene conversion from an inverted segmental duplication

Ehrlichia ruminantium is a tick-borne intracellular pathogen of ruminants that causes heartwater, a disease present in Sub-saharan Africa, islands in the Indian Ocean and the Caribbean, inducing significant economic losses. At present, three avirulent strains of E. ruminantium (Gardel, Welgevonden and Senegal isolates) have been produced by a process of serial passaging in mammalian cells in vitro, but unfortunately their use as vaccines do not offer a large range of protection against other strains, possibly due to the genetic diversity present within the species (Cangi et al. 2016). So far no genetic basis for virulence attenuation has been identified in any E. ruminantium strain that could offer targets to facilitate vaccine production. Virulence attenuated Senegal strains have been produced twice independently, and require many fewer passages to attenuate than the other strains. We compared the genomes of a virulent and attenuated Senegal strain and identified a likely attenuator gene, ntrX, a global transcription regulator and member of a two-component system that is linked to environmental sensing. This gene has an inverted partial duplicate close to the parental gene that shows evidence of gene conversion in different E. ruminantium strains. The pseudogenisation of the gene in the avirulent Senegal strain occurred by gene conversion from the duplicate to the parent, transferring a 4bp deletion which is unique to the Senegal strain partial duplicate amongst the wild isolates. We confirmed that the ntrX gene is not expressed in the avirulent Senegal strain by RT-PCR. The inverted duplicate structure combined with the 4bp deletion in the Senegal strain can explain both the attenuation and the faster speed of attenuation in the Senegal strain relative to other strains of E. ruminantium. Our results identify nrtX as a promising target for the generation of attenuated strains of E. ruminantium by random or directed mutagenesis that could be used for vaccine production.

genomics↗

bioRxiv2020

Different evolutionary pathways of HIV-1 between fetus and mother perinatal transmission pairs indicate unique immune selection pressure in fetuses

Study of evolution and selection pressure on HIV-1 in fetuses will lead to a better understanding of the role of immune responses in shaping virus evolution and vertical transmission. Detailed genetic analyses of HIV-1 env gene from 12 in utero transmission pairs show that most infections (67%) occur within two months from childbirth. In addition, env sequences from long-term infected fetuses were highly divergent and formed separate phylogenetic lineages from their cognate maternal viruses. Host selection sites unique to infant viruses were identified in regions frequently targeted by broadly neutralizing antibodies and T cell immune responses. The identification of unique selection sites in the env gene of fetal viruses indicates that the immune system in fetuses is capable of exerting selection pressure on viral evolution. Studying selection and evolution of HIV-1 or other viruses in fetuses can be an alternative approach to investigate adaptive immunity in fetuses.

microbiology↗

bioRxiv2020

A highly diverged ancient interface plays a key role in the origin and evolution of Methionine AdenosylTransferase superfamily

Methionine adenosyltransferase (MAT), which catalyzes the biosynthesis of S-adenosylmethionine from L-methionine and ATP, is an ancient, highly conserved enzyme present in all three domains of life. Although the MAT enzymes of each domain are believed to share a common ancestor, the sequences of archaeal MATs show a high degree of divergence from the sequences of bacterial and eukaryotic MATs. However, the structural and functional consequences of this sequence divergence are not well understood. Here, we use structural bioinformatics analysis and ancestral sequence reconstruction to highlight the consequences of archaeal MAT divergence. We show that the dimer interface containing the active site, which would be expected to be well conserved across all three domains, diverged considerably between the bacterial/eukaryotic MATs and archaeal MATs. Furthermore, the characterization of reconstructed ancestral archaeal MATs showed that they probably had low substrate specificity which expanded during their evolutionary trajectory hinting towards the observation that all the modern day MAT enzymes from the three-kingdom probably originated from a common specific ancestor and then archaea MATs diverged in sequence, structure and substrate specificity. Altogether, our results show that the archaea MAT is an ideal system for studying an enzyme family which evolved to display high degrees of divergence at the sequence/structural levels and yet are capable of performing the same catalytic reactions as their orthologous counterparts.

evolutionary biology↗

bioRxiv2020

Epigenetic regulator BMI1 promotes fusion-positive rhabdomyosarcoma proliferation and constitutes a novel therapeutic target

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma that continues to present significant challenges to pediatric oncology. There are two major subtypes of pediatric rhabdomyosarcoma, alveolar and embryonal. Alveolar rhabdomyosarcomas are characterized by the presence of a PAX-FOXO1 fusion protein and termed fusion-positive (FP-RMS); embryonal rhabdomyosarcomas (ERMS) lack these fusions and are termed fusion-negative (FN-RMS). Fusion-positive rhabdomyosarcoma (FP-RMS) harbors PAX-FOXO1 fusion proteins and has a worse overall outcome compared to FN-RMS, underscoring the critical need to identify novel targets for this disease. While fusion proteins remain challenging therapeutic targets, recent studies have begun to reveal the key intersection of PAX-FOXO1 fusion proteins with the malignant epigenome, suggesting that epigenetic proteins may serve as novel targets in FP-RMS. Here, we investigate the contribution of the epigenetic regulator BMI1 to FP-RMS. We examined RNA-seq tumor datasets and determined that BMI1 is robustly expressed in FP-RMS tumors, patient derived xenografts (PDXs), and cell lines. We depleted BMI1 using RNA interference and find that this leads to a marked decrease in cell growth. Next, we used two BMI1 inhibitors, PTC-209 and PTC-028, and showed that BMI1 inhibition decreases cell cycle progression and increases apoptosis in FP-RMS cell lines. In the in vivo setting, targeting BMI1 leads to decreased tumor growth. Mechanistically, we observe that BMI1 inhibition activates the tumor suppressive Hippo pathway. Collectively, these results identify BMI1 as a novel therapeutic vulnerability in FP-RMS and provide a foundation for further investigation of BMI1 in both FP-RMS and additional sarcoma histotypes.

cancer biology↗

bioRxiv2020

Single-cell transcriptomic profile reveals macrophage heterogeneity in medulloblastoma and their treatment-dependent recruitment

The role of macrophages in medulloblastoma, the most common malignant pediatric brain tumor, is unclear. Using single-cell RNA sequencing in a mouse model of sonic hedgehog medulloblastoma and analysis of bulk RNA sequencing of human medulloblastoma, we investigated macrophage heterogeneity. Our findings reveal differential recruitment of macrophages with molecular-targeted versus radiation therapy and identify an immunosuppressive monocyte-derived macrophages following radiation treatment of mouse medulloblastoma, uncovering potential strategies for immunomodulation as adjunctive therapy.

cancer biology↗

bioRxiv2019

Definition of alleles and altered regulatory motifs across Cas9-edited cell populations

BackgroundGenetic alteration of candidate response elements at their native chromosomal loci is the only valid determinant of their potential transcriptional regulatory activities. Targeted DNA cleavage by Cas9 coupled with cellular repair processes can produce arrays of alleles that can be defined by massively parallel sequencing by synthesis (SBS), presenting an opportunity to generate and survey edited cell populations that include informative alterations. Such editing efforts commonly rely on subclonal enrichment to isolate cells with preferred genotypic properties at target loci; short nucleotide adducts (indices/barcodes) allow PCR-amplified molecules from diverse sample sources to be pooled, sequenced, and demultiplexed to resolve source-specific content. Not widely available, however, are capabilities for barcoding thousands of clones, or for automated analysis of individual candidate regulatory loci PCR-amplified and sequenced from a genetically heterogeneous population--specifically, imputation of discrete genotype(s) by allele definition and abundance, and identification of altered regulatory factor binding motifs.\n\nResultsWe describe a panel of 192 8-nucleotide barcode primers compatible with Illumina(R) sequencing platforms, and the application of these barcodes to genotypic analysis of Cas9-edited clones. Permutations of the ninety-six i7 (read 1) and ninety-six i5 (read 2) barcodes allow unique labeling of up to 9,216 distinct samples. We created three independent Python scripts: SampleSheet.py automates construction of Illumina(R) Sample Sheets encoding up to 9,216 barcode:sample relationships; ImputedGenotypes.py defines alleles and imputes genotypes from demultiplexed fastq files; CollatedMotifs.py flags transcription factor recognition motif matches altered in alleles relative to a reference sequence.\n\nConclusionsCode-enabled definition of alleles and regulatory motifs in sequenced, demultiplexed amplicons facilitates evaluation of genetic diversity in up to 9,216 distinct samples. Here, we demonstrate the utility of three scripts in analysis of cell populations targeted by Cas9 for disruption of glucocorticoid receptor (GR) binding sites near FKBP5, a GR-regulated gene in the human adenocarcinoma cell line A549. SampleSheet.py, ImputedGenotypes.py, and CollatedMotifs.py operate independently and are broadly applicable beyond the case described here.

molecular biology↗