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José Javier Morales-Núñez,1 Mariel García-Chagollán,1 José Francisco Muñoz-Valle,1 Saúl Alberto Díaz-Pérez,1 Paola Carolina Torres-Hernández,2 Saraí Citlalic Rodríguez-Reyes,3 Guillermo Santoscoy-Ascencio,4 José Julio Sierra García de Quevedo,4 Jorge Hernández-Bello1 1Institute of Research in Biomedical Sciences, University Center of Health Sciences (CUCS), University of Guadalajara, Guadalajara, Jalisco, Mexico; 2Immunology Laboratory, University Center of Health Sciences (CUCS), University of Guadalajara, Guadalajara, Jalisco, Mexico; 3Institute of Translational Nutrigenetics and Nutrigenomics, University Center of Health Sciences (CUCS), University of Guadalajara, Guadalajara, Jalisco, Mexico; 4Unidad de Patología Clinica (UPC), Guadalajara, Jalisco, MexicoCorrespondence: Jorge Hernández-Bello,s Institute of Research in Biomedical Sciences, University Center of Health Sciences (CUCS), University of Guadalajara, Guadalajara, Jalisco, 44340, Mexico, Tel +52 3334509355, Email jorge.hernandezbello@cucs.udg.mxPurpose: Understanding the humoral immune response dynamics carried out by B cells in COVID-19 vaccination is little explored; therefore, we analyze the changes induced in the different cellular subpopulations of B cells after vaccination with BNT162b2 (Pfizer-BioNTech).Methods: This prospective cohort study evaluated thirty-nine immunized health workers (22 with prior COVID-19 and 17 without prior COVID-19) and ten subjects not vaccinated against SARS-CoV-2 (control group). B cell subpopulations (transitional, mature, naïve, memory, plasmablasts, early plasmablast, and double-negative B cells) and neutralizing antibody levels were analyzed and quantified by flow cytometry and ELISA, respectively.Results: The dynamics of the B cells subpopulations after vaccination showed the following pattern: the percentage of transitional B cells was higher in the prior COVID-19 group (p < 0.05), whereas virgin B cells were more prevalent in the group without prior COVID-19 (p < 0.05), mature B cells predominated in both vaccinated groups (p < 0.01), and memory B cells, plasmablasts, early plasmablasts, and double-negative B cells were higher in the not vaccinated group (p < 0.05).Conclusion: BNT162b2 vaccine induces changes in B cell subpopulations, especially generating plasma cells and producing neutralizing antibodies against SARS-CoV-2. However, the previous infection with SARS-CoV-2 does not significantly alter the dynamics of these subpopulations but induces more rapid and optimal antibody production.Keywords: B cell, BNT162b2, SARS-CoV-2, immunophenotype, vaccine

Early in mammalian female embryonic development, an unidentiffed developmental signal causes heterochromatin to form along nearly the entire length of one X chromosome. We generated Xist knockout mice and showed that the X-linked Xist locus is required in cis for X-inactivation in the extraembryonic tissue of females (Marahrens et al., 1997). Analysis of the somatic cells of mutant females indicated that the Xist mutation caused primary nonrandom X-inactivation [Marahrens et al., 1998). This was confirmed by following the expression of an X-linked GFP gene through early mouse development. We proposed that (1) the two Xist loci in female cells physically associate with one another resulting in one Xist allele being remodeled into heterochromatin and (2) that the heterochromatic Xist locus physically interacts with heterochromatic LINE elements dispersed throughout the X chromosome stimulating heterochromatin to spread from LINE sequence outward into unique sequence until the entire X chromosome is transcriptionally inactivated (Marahrens, 1999). Xist-LINE interactions may allow the Xist RNA, which is required for heterochromatin formation, to be transferred to distant chromosomal regions. More recently, we have been examining the phenotype caused by a Xist deletion on the active X chromosome. At the chromosomal level we have been examining the replication timing, stability' of gene silencing, and chromatin structure. We have also been characterizing the phenotype of embryos of Xist+/- mothers. Our results indicate that a Xist deletion in the nontranscribed allele of the active X chromosome causes the chromatin structure throughout the entire inactive X chromosome to be altered. This appears to result in a destabilization of gene silencing in adult females resulting in growth retardation of their embryos when they are pregnant. Our results can only be explained by a transvection-based mechanism.

Discusses a study on the role of molecular methods in evaluating biological water treatment processes. Significant development occurring in research and biological treatment processes for wastewater and other wastes; Areas for use of molecular tools in a wastewater treatment plant; Effect of temperature on performance of thermophilic digesters at the Hyperion Treatment Plant.

Human diseases may be modeled in animals to allow preclinical assessment of putative new clinical interventions. Recent, highly publicized failures of large clinical trials called into question the rigor, design, and value of preclinical assessment. We established the Stroke Preclinical Assessment Network (SPAN) to design and implement a randomized, controlled, blinded, multi-laboratory trial for the rigorous assessment of candidate stroke treatments combined with intravascular thrombectomy. Efficacy and futility boundaries in a multi-arm multi-stage statistical design aimed to exclude from further study highly effective or futile interventions after each of four sequential stages. Six independent research laboratories performed a standard focal cerebral ischemic insult in five animal models that included equal numbers of males and females: young mice, young rats, aging mice, mice with diet-induced obesity, and spontaneously hypertensive rats. The laboratories adhered to a common protocol and efficiently enrolled 2615 animals with full data completion and comprehensive animal tracking. SPAN successfully implemented treatment masking, randomization, prerandomization inclusion and exclusion criteria, and blinded assessment of outcomes. The SPAN design and infrastructure provide an effective approach that could be used in similar preclinical, multi-laboratory studies in other disease areas and should help improve reproducibility in translational science.

Background: Radiotherapy impacts the local immune response to cancers. Prostate Stereotactic Body Radiotherapy (SBRT) is a highly focused method to deliver radiotherapy often used to treat prostate cancer. This is the first direct comparison of immune cells within prostate cancers before and after SBRT in patients., Methods: Prostate cancers before and 2 weeks after SBRT are interrogated by multiplex immune fluorescence targeting various T cells and macrophages markers and analyzed by cell and pixel density, as part of a clinical trial of SBRT neoadjuvant to radical prostatectomy., Results: Two weeks after SBRT, CD68, and CD163 macrophages are significantly increased while CD8 T cells are decreased. SBRT markedly alters the immune environment within prostate cancers., (© 2022. The Author(s).)

Cerebral ischemia and reperfusion initiate cellular events in brain that lead to neurological disability. Investigating these cellular events provides ample targets for developing new treatments. Despite considerable work, no such therapy has translated into successful stroke treatment. Among other issues-such as incomplete mechanistic knowledge and faulty clinical trial design-a key contributor to prior translational failures may be insufficient scientific rigor during preclinical assessment: nonblinded outcome assessment; missing randomization; inappropriate sample sizes; and preclinical assessments in young male animals that ignore relevant biological variables, such as age, sex, and relevant comorbid diseases. Promising results are rarely replicated in multiple laboratories. We sought to address some of these issues with rigorous assessment of candidate treatments across 6 independent research laboratories. The Stroke Preclinical Assessment Network (SPAN) implements state-of-the-art experimental design to test the hypothesis that rigorous preclinical assessment can successfully reduce or eliminate common sources of bias in choosing treatments for evaluation in clinical studies. SPAN is a randomized, placebo-controlled, blinded, multilaboratory trial using a multi-arm multi-stage protocol to select one or more putative stroke treatments with an implied high likelihood of success in human clinical stroke trials. The first stage of SPAN implemented procedural standardization and experimental rigor. All participating research laboratories performed middle cerebral artery occlusion surgery adhering to a common protocol and rapidly enrolled 913 mice in the first of 4 planned stages with excellent protocol adherence, remarkable data completion and low rates of subject loss. SPAN stage 1 successfully implemented treatment masking, randomization, prerandomization inclusion/exclusion criteria, and blinded assessment to exclude bias. Our data suggest that a large, multilaboratory, preclinical assessment effort to reduce known sources of bias is feasible and practical. Subsequent SPAN stages will evaluate candidate treatments for potential success in future stroke clinical trials using aged animals and animals with comorbid conditions.

Background: Hundreds of ongoing clinical trials combine radiation therapy, mostly delivered as stereotactic body radiotherapy (SBRT), with immune checkpoint blockade. However, our understanding of the effect of radiotherapy on the intratumoral immune balance is inadequate, hindering the optimal design of trials that combine radiation therapy with immunotherapy. Our objective was to characterize the intratumoral immune balance of the malignant prostate after SBRT in patients., Methods: Sixteen patients with high-risk, non-metastatic prostate cancer at comparable Gleason Grade disease underwent radical prostatectomy with (n = 9) or without (n = 7) neoadjuvant SBRT delivered in three fractions of 8 Gy over 5 days completed 2 weeks before surgery. Freshly resected prostate specimens were processed to obtain single-cell suspensions, and immune-phenotyped for major lymphoid and myeloid cell subsets by staining with two separate 14-antibody panels and multicolor flow cytometry analysis., Results: Malignant prostates 2 weeks after SBRT had an immune infiltrate dominated by myeloid cells, whereas malignant prostates without preoperative treatment were more lymphoid-biased (myeloid CD45 + cells 48.4 ± 19.7% vs. 25.4 ± 7.0%; adjusted p-value = 0.11; and CD45 + lymphocytes 51.6 ± 19.7% vs. 74.5 ± 7.0%; p = 0.11; CD3 + T cells 35.2 ± 23.8% vs. 60.9 ± 9.7%; p = 0.12; mean ± SD)., Conclusion: SBRT drives a significant lymphoid to myeloid shift in the prostate-tumor immune infiltrate. This may be of interest when combining SBRT with immunotherapies, particularly in prostate cancer.

Purpose: This study aimed to evaluate the feasibility and safety of prostate stereotactic body radiation therapy (SBRT) neoadjuvant to radical prostatectomy (RP) in a phase 1 trial. The primary endpoint was treatment completion rate without severe acute surgical complications. Secondary endpoints included patient-reported quality of life and physician-reported toxicities., Methods and Materials: Patients with nonmetastatic high-risk or locally advanced prostate cancer received 24 Gy in 3 fractions to the prostate and seminal vesicles over 5 days, completed 2 weeks before RP. Patients with pN1 disease were treated after multidisciplinary discussion and shared decision making. Patient-reported quality of life (International Prostate Symptom Score and Expanded Prostate Cancer Index Composite 26-item version questionnaires) and physician-reported toxicity (Common Terminology Criteria for Adverse Events, version 4.03) were assessed before SBRT, immediately before surgery, and at 3-month intervals for 1 year., Results: Twelve patients were enrolled, and 11 completed treatment (1 patient had advanced disease on prostate-specific membrane antigen positron emission tomography after enrollment but before treatment). There were no significant surgical complications. After RP, 2 patients underwent additional radiation therapy to nodes with androgen suppression for pN1 disease. Median follow-up after completion of treatment was 20.1 months, with 9 of 11 patients having a follow-up period of >12 months. Two patients had biochemical recurrence (prostate-specific antigen ≥0.05) within the first 12 months, with an additional 2 patients found to have biochemical recurrence after the 12-month period. The highest Common Terminology Criteria for Adverse Events genitourinary grades were 0, 1, 2, and 3 (n = 1, 4, 4, and 2, respectively), and the highest gastrointestinal grades were 0, 1, and 2 (n = 9, 1, and 1, respectively). At 12 months, incontinence was the only grade ≥2 toxicity. One and 2 of 9 patients had grade 2 and 3 incontinence, respectively. On the Expanded Prostate Cancer Index Composite (26-item version), the mean/median changes in scores from baseline to 12 months were -32.8/-31.1 for urinary incontinence, -1.6/-6.2 for urinary irritative/obstructive, -2.1/0 for bowel, -34.4/-37.5 for sexual function, and -10.6/-2.5 for hormonal. The mean/median change in International Prostate Symptom Score from baseline to 12 months was 0.5/0.5., Conclusions: RP after neoadjuvant SBRT appears to be feasible and safe at the dose tested. The severity of urinary incontinence may be higher than RP alone., (Published by Elsevier Inc.)

Pyrrole-imidazole (Py-Im) polyamides are synthetic non-genotoxic minor groove-binding small molecules. We hypothesized that Py-Im polyamides can modulate the cellular response to ionizing radiation. Pre-treatment of cells with a Py-Im polyamide prior to exposure to ionizing radiation resulted in a delay in resolution of phosphorylated γ-H2AX foci, increase in XRCC1 foci, and reduced cellular replication potential. RNA-sequencing of cell lines exposed to the polyamide showed induction of genes related to the ultraviolet radiation response. We observed that the polyamide is almost 10-fold more toxic to a cell line deficient in DNA ligase 3 as compared to the parental cell line. Alkaline single cell gel electrophoresis reveals that the polyamide induces genomic fragmentation in the ligase 3 deficient cell line but not the corresponding parental line. The polyamide interferes directly with DNA ligation in vitro. We conclude that Py-Im polyamides may be further explored as sensitizers to genotoxic therapies.

H3N2 viruses continuously acquire mutations in the hemagglutinin (HA) glycoprotein that abrogate binding of human antibodies. During the 2014-2015 influenza season, clade 3C.2a H3N2 viruses possessing a new predicted glycosylation site in antigenic site B of HA emerged, and these viruses remain prevalent today. The 2016-2017 seasonal influenza vaccine was updated to include a clade 3C.2a H3N2 strain; however, the egg-adapted version of this viral strain lacks the new putative glycosylation site. Here, we biochemically demonstrate that the HA antigenic site B of circulating clade 3C.2a viruses is glycosylated. We show that antibodies elicited in ferrets and humans exposed to the egg-adapted 2016-2017 H3N2 vaccine strain poorly neutralize a glycosylated clade 3C.2a H3N2 virus. Importantly, antibodies elicited in ferrets infected with the current circulating H3N2 viral strain (that possesses the glycosylation site) and humans vaccinated with baculovirus-expressed H3 antigens (that possess the glycosylation site motif) were able to efficiently recognize a glycosylated clade 3C.2a H3N2 virus. We propose that differences in glycosylation between H3N2 egg-adapted vaccines and circulating strains likely contributed to reduced vaccine effectiveness during the 2016-2017 influenza season. Furthermore, our data suggest that influenza virus antigens prepared via systems not reliant on egg adaptations are more likely to elicit protective antibody responses that are not affected by glycosylation of antigenic site B of H3N2 HA., Competing Interests: Conflict of interest statement: S.J.Z., K.P., M.E.G., K.K., S.D.P., P.C.W., A.J.S., S.C., and S.E.H. have no conflicts of interest. J.J.T. is an advisor (nonpaid) for Protein Sciences and is on the scientific advisory board or received consulting payments for Sequiris, Medicago, Takeda, and Flugen. J.J.T.’s laboratory has also received support from Sanofi., (Copyright © 2017 the Author(s). Published by PNAS.)

The N-end rule pathway of protein degradation targets proteins with destabilizing N-terminal residues. Ubr2 is one of the E3 ubiquitin ligases of the mouse N-end rule pathway. We have previously shown that Ubr2-/- male mice are infertile, owing to the arrest of spermatocytes between the leptotene/zygotene and pachytene of meiosis I, the failure of chromosome pairing, and subsequent apoptosis. Here, we report that mouse fibroblast cells derived from Ubr2-/- embryos display genome instability. The frequency of chromosomal bridges and micronuclei were much higher in Ubr2-/- fibroblasts than in +/+ controls. Metaphase chromosome spreads from Ubr2-/- cells revealed a high incidence of spontaneous chromosomal gaps, indicating chromosomal fragility. These fragile sites were generally replicated late in S phase. Ubr2-/- cells were hypersensitive to mitomycin C, a DNA cross-linking agent, but displayed normal sensitivity to gamma-irradiation. A reporter assay showed that Ubr2-/- cells are significantly impaired in the homologous recombination repair of a double strand break. In contrast, Ubr2-/- cells appeared normal in an assay for non-homologous end joining. Our results therefore unveil the role of the ubiquitin ligase Ubr2 in maintaining genome integrity and in homologous recombination repair.

ATM and ATR are well documented for their roles in maintaining the integrity of genomic DNA by responding to DNA damage and preparing the cell for repair. Since ATM and ATR have been reported to exist in complexes with histone deacetylases, we asked whether Atm and Atr might also uphold gene silencing by heterochromatin. We show that the Atm/Atr inhibitor 2-aminopurine causes the inactive X chromosome to accumulate abnormal chromatin and undergo unwanted gene reactivation. We provide evidence that this gene expression from the inactive X chromosome is not a byproduct of the accumulation of DNA breaks. Individually inhibiting Atm and Atr by either small interfering RNA or the expression of dominant-negative ATM and ATR constructs also compromised X-inactivation. Atm and Atr, therefore, not only function in responding to DNA damage but perhaps also are involved in gene silencing via the maintenance of heterochromatin.

In female mammalian cells, the inactive X chromosome is replicated late in S phase while the active X chromosome is replicated earlier. The replication times of the X chromosomes reflect a general trend in which late replication is associated with gene repression and earlier replication with transcriptional competence. The X-linked Xist gene is expressed exclusively from the inactive X chromosome where it is involved in the initiation and maintenance of X-inactivation. In contrast, no biological activity has been assigned to the Xist locus of the active X chromosome where the Xist gene is transcriptionally silenced. Here, we provide evidence that the element(s) at the nontranscribed Xist locus of the active X chromosome controls chromosomal replication timing in cis.

We have used a variety of methods to characterize the genome of the archaeon Methanosarcina thermophila TM-1. Pulsed-field gel analysis indicates a genome size of 2.8 Mb. We have constructed a bacterial artificial chromosome (BAC) library of M. thermophila and have used it to generate physical maps for this organism. The library is made up of 384 clones with an average insert size of 58 kb representing 8.0 genome equivalents. The utility of the library for low-resolution physical mapping was shown by identifying NotI linking clones and using these to order the NotI macrorestriction fragments of M. thermophila into a 2.8 Mb map. Hybridization of nine single copy genes and a 16S rRNA sequence to these macrorestriction fragments forms the basis for the first genetic map in this organism. High-resolution physical maps, consisting of overlapping clones, have been created using HindIII fingerprints of BAC clones. In this way, we identified a minimal path of five clones that span a 270 kb NotI fragment. The ease of manipulating BAC clones makes the BAC system an excellent choice for the construction of low-resolution and high-resolution physical and genetic maps of archaeal genomes. It also provides a substrate for future genome-sequencing efforts.