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Aviram N, Thornal AN, Zeevi D, Marraffini LA
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Different modes of spacer acquisition by the Staphylococcus epidermidis type III-A CRISPR-Cas system (opens in new window)

NUCLEIC ACIDS RESEARCH 2022 FEB 22; 50(3):1661-1672
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CRISPR-Cas systems provide prokaryotic organisms with an adaptive defense mechanism that acquires immunological memories of infections. This is accomplished by integration of short fragments from the genome of invaders such as phages and plasmids, called 'spacers', into the CRISPR locus of the host. Depending on their genetic composition, CRISPR-Cas systems can be classified into six types, I-VI, however spacer acquisition has been extensively studied only in type I and II systems. Here, we used an inducible spacer acquisition assay to study this process in the type III-A CRISPR-Cas system of Staphylococcus epidermidis, in the absence of phage selection. Similarly to type I and II spacer acquisition, this type III system uses Cas1 and Cas2 to preferentially integrate spacers from the chromosomal terminus and free dsDNA ends produced after DNA breaks, in a manner that is enhanced by the AddDNA repair complex. Surprisingly, a different mode of spacer acquisition from rRNA and tRNA loci, which spans only the transcribed sequences of these genes and is not enhanced by AddAB, was also detected. Therefore, our findings reveal both common mechanistic principles that may be conserved in all CRISPR-Cas systems, as well as unique and intriguing features of type III spacer acquisition.
Saito Y, Hawley BR, Puno MR, Sarathy SN, Lima CD, Jaffrey SR, Darnell RB, Keeney S, Jain D
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YTHDC2 control of gametogenesis requires helicase activity but not m(6)A binding (opens in new window)

GENES & DEVELOPMENT 2022 FEB 1; 36(3-4):180-194
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In this study, Saito et al. sought to understand how the N-6-methyladenosine (m(6)A) reader and RNA helicase YTHDC2 switches cells from mitotic to meiotic gene expression programs. Their findings provide insight into YTHDC2's mechanism, and they propose a model in which YTHDC2 binds transcripts independent of m(6)A status and regulates gene expression during multiple stages of meiosis by distinct mechanisms. Mechanisms regulating meiotic progression in mammals are poorly understood. The N-6-methyladenosine (m(6)A) reader and 3 ' -> 5 ' RNA helicase YTHDC2 switches cells from mitotic to meiotic gene expression programs and is essential for meiotic entry, but how this critical cell fate change is accomplished is unknown. Here, we provide insight into its mechanism and implicate YTHDC2 in having a broad role in gene regulation during multiple meiotic stages. Unexpectedly, mutation of the m(6)A-binding pocket of YTHDC2 had no detectable effect on gametogenesis and mouse fertility, suggesting that YTHDC2 function is m(6)A-independent. Supporting this conclusion, CLIP data defined YTHDC2-binding sites on mRNA as U-rich and UG-rich motif-containing regions within 3 ' UTRs and coding sequences, distinct from the sites that contain m(6)A during spermatogenesis. Complete loss of YTHDC2 during meiotic entry did not substantially alter translation of its mRNA binding targets in whole-testis ribosome profiling assays but did modestly affect their steady-state levels. Mutation of the ATPase motif in the helicase domain of YTHDC2 did not affect meiotic entry, but it blocked meiotic prophase I progression, causing sterility. Our findings inform a model in which YTHDC2 binds transcripts independent of m(6)A status and regulates gene expression during multiple stages of meiosis by distinct mechanisms.
Schmidt F, Muecksch F, Weisblum Y, Da Silva J, Bednarski E, Cho AL, Wang ZJ, Gaebler C, Caskey M, Nussenzweig MC, Hatziioannou T, Bieniasz PD
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Plasma Neutralization of the SARS-CoV-2 Omicron Variant (opens in new window)

NEW ENGLAND JOURNAL OF MEDICINE 2022 FEB 10; 386(6):599-601
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Omicron Neutralization after the Third Vaccine Dose Neutralization assays of SARS-CoV-2 pseudoviruses expressing wild-type, omicron, or a synthetic resistant spike protein in plasma drawn from 47 people over time showed much lower omicron neutralization than Wuhan-hu-1 neutralization after two doses of an mRNA vaccine. However, samples from persons vaccinated after recovery from Covid-19 and those who received a booster vaccine had high levels of omicron neutralization.
Goel S, Kuehn HS, Chinen J, Niemela J, Stoddard J, Yamanaka D, Garofalo M, Samir S, Migaud M, Oikonomou V, Fleisher T, Puel A, Lionakis MS, Rosenzweig SD
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CARD9 Expression Pattern, Gene Dosage, and Immunodeficiency Phenotype Revisited (opens in new window)

JOURNAL OF CLINICAL IMMUNOLOGY 2022 FEB; 42(2):336-349
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Background CARD9 deficiency is an autosomal recessive primary immunodeficiency underlying increased susceptibility to fungal infection primarily presenting as invasive CNS Candida and/or cutaneous/invasive dermatophyte infections. More recently, a rare heterozygous dominant negative CARD9 variant c.1434 + 1G> C was reported to be protective from inflammatory bowel disease. Objective We studied two siblings carrying homozygous CARD9 variants (c.1434 + 1G> C) and born to heterozygous asymptomatic parents. One sibling was asymptomatic and the other presented with candida esophagitis, upper respiratory infections, hypogammaglobulinemia, and low class-switched memory B cells. Methods and Results The CARD9 c.1434 + 1G > C variant generated two mutant transcripts confirmed by mRNA and protein expression: an out-of-frame c.1358-1434 deletion/ similar to 55 kDa protein (CARD9 Delta ex.11) and an in-frame c.1417-1434 deletion/ similar to 61 kDa protein (CARD9 Delta 18 nt.). Neither transcript was able to form a complete/functional CBM complex, which includes TRIM62. Based on the index patient's CVID-like phenotype, CARD9 expression was tested and detected in lymphocytes and monocytes from humans and mice. The functional impact of different CARD9 mutations and gene dosage conditions was evaluated in heterozygous and homozygous c.1434 +1 G> C members of the index family, and in WT (two WT alleles), haploinsufficiency (one WT, one null allele), and null (two null alleles) individuals. CARD9 gene dosage impacted lymphocyte and monocyte functions including cytokine generation, MAPK activation, T-helper commitment, transcription, plasmablast differentiation, and immunoglobulin production in a differential manner. Conclusions CARD9 exon 11 integrity is critical to CBM complex function. CARD9 is expressed and affects particular T and B cell functions in a gene dosage-dependent manner, which in turn may contribute to the phenotype of CARD9 deficiency.
Monaco H, Liu KS, Sereno T, Deforet M, Taylor BP, Chen YY, Reagor CC, Xavier JB
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Spatial-temporal dynamics of a microbial cooperative behavior resistant to cheating (opens in new window)

NATURE COMMUNICATIONS 2022 FEB 7; 13(1):? Article 721
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Much of our understanding of bacterial behavior stems from studies in liquid culture. In nature, however, bacteria frequently live in densely packed spatially-structured communities. How does spatial structure affect bacterial cooperative behaviors? In this work, we examine rhamnolipid production-a cooperative and virulent behavior of Pseudomonas aeruginosa. Here we show that, in striking contrast to well-mixed liquid culture, rhamnolipid gene expression in spatially-structured colonies is strongly associated with colony specific growth rate, and is impacted by perturbation with diffusible quorum signals. To interpret these findings, we construct a data-driven statistical inference model which captures a length-scale of bacterial interaction that develops over time. Finally, we find that perturbation of P. aeruginosa swarms with quorum signals preserves the cooperating genotype in competition, rather than creating opportunities for cheaters. Overall, our data demonstrate that the complex response to spatial localization is key to preserving bacterial cooperative behaviors. Bacteria often live in densely packed, spatially-structured communities; however, much of our understanding of their behavior stems from studies in liquid culture. Here, Monaco et al. show how spatial structure and quorum sensing modulate a cooperative behavior in colonies of Pseudomonas aeruginosa.
Patrizio P, Albertini DF, Gleicher N, Caplan A
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The changing world of IVF: the pros and cons of new business models offering assisted reproductive technologies (vol 20, pg 1, 2022) (opens in new window)

JOURNAL OF ASSISTED REPRODUCTION AND GENETICS 2022 FEB; 39(2):315-315
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Moldwin RM, Nursey V, Yaskiv O, Dalvi S, Macdonald EJ, Funaro M, Zhang CL, DeGouveia W, Ruzimovsky M, Rilo HR, Miller EJ, Najjar S, Tabansky I, Stern JNH
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Immune cell profiles of patients with interstitial cystitis/bladder pain syndrome (opens in new window)

JOURNAL OF TRANSLATIONAL MEDICINE 2022 FEB 21; 20(1):? Article 97
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Interstitial cystitis/bladder pain syndrome (IC/BPS) is a disorder characterized by bladder pain upon filling which severely affects quality of life. Clinical presentation can vary. Local inflammatory events typify the clinical presentation of IC/BPS patients with Hunner lesions (IC/BPS-HL). It has previously been proposed that B cells are more prevalent in HL, but understanding their exact role in this environment requires a more complete immunological profile of HL. We characterized immunological dysfunction specifically in HL using immunohistochemistry. We detected significantly more plasma cells (50x increase, p < 0.0001), B cells (28x increase, p < 0.0001), T cells (3x increase, p < 0.0001), monocytes/macrophages (6x increase, p < 0.0001), granulocytes (4x increase, p < 0.0001), and natural killer cells (2x increase, p = 0.0249) in IC/BPS patients with HL than in unaffected controls (UC). Patients with IC/BPS-HL also had significantly elevated urinary levels of IL-6 (p = 0.0054), TNF-alpha (p = 0.0064) and IL-13 (p = 0.0304) compared to patients with IC/BPS without HL (IC/BPS-NHL). In contrast, IL-12p70 levels were significantly lower in the patients with HL than in those without these lesions (p = 0.0422). Different cytokines were elevated in the urine of IC/BPS patients with and without HL, indicating that different disease processes are active in IC/BPS patients with and without HL. Elevated levels of CD138+, CD20+, and CD3+ cells in HL are consistent B and T-cell involvement in disease processes within HL.
Kaczmarek E, Pyman B, Nanayakkara J, Tuschl T, Tyryshkin K, Renwick N, Mousavi P
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Discriminating Neoplastic from Nonneoplastic Tissues Using an miRNA-Based Deep Cancer Classifier (opens in new window)

AMERICAN JOURNAL OF PATHOLOGY 2022 FEB; 192(2):344-352
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Next-generation sequencing has enabled the collection of large biological data sets, allowing novel molecular-based classification methods to be developed for increased understanding of disease. miRNAs are small regulatory RNA molecules that can be quantified using next-generation sequencing and are excellent classificatory markers. Herein, a deep cancer classifier (DCC) was adapted to differentiate neoplastic from nonneoplastic samples using comprehensive miRNA expression profiles from 1031 human breast and skin tissue samples. The classifier was fine-tuned and evaluated using 750 neoplastic and 281 nonneoplastic breast and skin tissue samples. Performance of the DCC was compared with two machine-learning classifiers: support vector machine and random forests. In addition, performance of feature extraction through the DCC was also compared with a developed feature selection algorithm, cancer specificity. The DCC had the highest performance of area under the receiver operating curve and high performance in both sensitivity and specificity, unlike machine-learning and feature selection models, which often performed well in one metric compared with the other. In particular, deep learning had noticeable advantages with highly heterogeneous data sets. In addition, our cancer specificity algorithm identified candidate biomarkers for differentiating neoplastic and nonneoplastic tissue samples (eg, miR-144 and miR-375 in breast cancer and miR-375 and miR-451 in skin cancer).
Calpena E, Wurmser M, McGowan SJ, Atique R, Bertola DR, Cunningham ML, Gustafson JA, Johnson D, Morton JEV, Passos-Bueno MR, Timberlake AT, Lifton RP, Wall SA, Twigg SRF, Maire P, Wilkie AOM
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Unexpected role of SIX1 variants in craniosynostosis: expanding the phenotype of SIX1-related disorders (opens in new window)

JOURNAL OF MEDICAL GENETICS 2022 FEB; 59(2):165-169
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Background Pathogenic heterozygous SIX1 variants (predominantly missense) occur in branchio-otic syndrome (BOS), but an association with craniosynostosis has not been reported. Methods We investigated probands with craniosynostosis of unknown cause using whole exome/genome (n=628) or RNA (n=386) sequencing, and performed targeted resequencing of SIX1 in 615 additional patients. Expression of SIX1 protein in embryonic cranial sutures was examined in the Six1 ( nLacZ/+ ) reporter mouse. Results From 1629 unrelated cases with craniosynostosis we identified seven different SIX1 variants (three missense, including two de novo mutations, and four nonsense, one of which was also present in an affected twin). Compared with population data, enrichment of SIX1 loss-of-function variants was highly significant (p=0.00003). All individuals with craniosynostosis had sagittal suture fusion; additionally four had bilambdoid synostosis. Associated BOS features were often attenuated; some carrier relatives appeared non-penetrant. SIX1 is expressed in a layer basal to the calvaria, likely corresponding to the dura mater, and in the mid-sagittal mesenchyme. Conclusion Craniosynostosis is associated with heterozygous SIX1 variants, with possible enrichment of loss-of-function variants compared with classical BOS. We recommend screening of SIX1 in craniosynostosis, particularly when sagittal +/- lambdoid synostosis and/or any BOS phenotypes are present. These findings highlight the role of SIX1 in cranial suture homeostasis.
Gleicher N, Darmon SK, Molinari E, Patrizio P, Barad DH
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Importance of IGF-I levels in IVF: potential relevance for growth hormone (GH) supplementation (opens in new window)

JOURNAL OF ASSISTED REPRODUCTION AND GENETICS 2022 FEB; 39(2):409-416
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Purpose Growth hormone (GH) supplementation in association with in vitro fertilization (IVF) is worldwide again increasing, even though study outcomes have been discrepant. Since GH acts via insulin-like growth factor-1 (IGF-1), its utilization in IVF would only seem to make sense with low IGF-1. We, therefore, determined whether IGF-I levels affect IVF outcomes. Methods Retrospectively, 302 consecutive first fresh, non-donor IVF cycles were studied, excluding patients on GH supplementation. Patients were divided into 3 subgroups: IGF-1 in lower 25th percentile (group A, < 132 ng/mL, n = 64); 25th-75th percentile (B, 133-202 ng/mL, n = 164), and upper 25th percentile (C, > 202 ng/mL, n = 74). IGF-1 was tested immunochemiluminometric with normal range at 78-270 ng/mL. Because of the study patients' adverse selection and low pregnancy chances, the main outcome measure for the study was cycle cancellation. Secondary outcomes were oocyte numbers, embryos transferred, pregnancies, and live births. Results Group A was significantly older than B and C (P = 0.019). IGF-1 decreased with increasing age per year by 2.2 +/- 0.65 ng/mL (P = 0.0007). FSH was best in group B and worst in A (trend, P = 0.085); AMH was best in B and worst in A (N.S.). Cycle cancellations were lowest in C (11.6%) and highest in A (25.0%; P = 0.042). This significance further improved with age adjustment (P = 0.021). Oocytes, embryo numbers, pregnancies, and live birth rates did not differ, though oocyte numbers trended highest in B. Conclusions Here presented results support the hypothesis that IGF-1 levels affect IVF outcomes. GH treatments, therefore, may be effective only with low IGF-1.