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Background: The treatment of head and neck tumors remains a challenge due to their reduced radiosensitivity. Small molecule kinase inhibitors (smKI) that inhibit the DNA damage response, may increase the radiosensitivity of tumor cells. However, little is known about how the immunophenotype of the tumor cells is modulated thereby. Therefore, we investigated whether the combination of ATM or ATR inhibitors with hypo-fractionated radiotherapy (RT) has a different impact on the expression of immune checkpoint markers (extrinsic), the release of cytokines or the transcriptome (intrinsic) of head and neck squamous cell carcinoma (HNSCC) cells., Methods: The toxic and immunogenic effects of the smKI AZD0156 (ATMi) and VE-822 (ATRi) in combination with a hypo-fractionated scheme of 2x5Gy RT on HPV-negative (HSC4, Cal-33) and HPV-positive (UM-SCC-47, UD-SCC-2) HNSCC cell lines were analyzed as follows: cell death (necrosis, apoptosis; detected by AnxV/PI), expression of immunostimulatory (ICOS-L, OX40-L, TNFSFR9, CD70) and immunosuppressive (PD-L1, PD-L2, HVEM) checkpoint marker using flow cytometry; the release of cytokines using multiplex ELISA and the gene expression of Cal-33 on mRNA level 48 h post-RT., Results: Cell death was mainly induced by the combination of RT with both inhibitors, but stronger with ATRi. Further, the immune phenotype of cancer cells, not dying from combination therapy itself, is altered predominantly by RT+ATRi in an immune-stimulatory manner by the up-regulation of ICOS-L. However, the analysis of secreted cytokines after treatment of HNSCC cell lines revealed an ambivalent influence of both inhibitors, as we observed the intensified secretion of IL-6 and IL-8 after RT+ATRi. These findings were confirmed by RNAseq analysis and further the stronger immune-suppressive character of RT+ATMi was enlightened. We detected the down-regulation of a central protein of cytoplasmatic sensing pathways of nucleic acids, RIG-1, and found one immune-suppressive target, EDIL3, strongly up-regulated by RT+ATMi., Conclusion: Independent of a restrictive toxicity, the combination of RT + either ATMi or ATRi leads to comprehensive and immune-modulating alterations in HNSCC. This includes pro-inflammatory signaling induced by RT + ATRi but also anti-inflammatory signals. These findings were confirmed by RNAseq analysis, which further highlighted the immune-suppressive nature of RT + ATMi., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Meidenbauer, Wachter, Schulz, Mostafa, Zülch, Frey, Fietkau, Gaipl and Jost.)

Autophagy supervises the proteostasis and survival of B lymphocytic cells. Trk-fused gene (TFG) promotes autophagosome-lysosome flux in murine CH12 B cells, as well as their survival. Hence, quantitative proteomics of CH12 tfg KO and WT B cells in combination with lysosomal inhibition should identify proteins that are prone to lysosomal degradation and contribute to autophagy and B cell survival. Lysosome inhibition via NH 4 Cl unexpectedly reduced a number of proteins but increased a large cluster of translational, ribosomal, and mitochondrial proteins, independent of TFG. Hence, we propose a role for lysosomes in ribophagy in B cells. TFG-regulated proteins include CD74, BCL10, or the immunoglobulin JCHAIN. Gene ontology (GO) analysis reveals that proteins regulated by TFG alone, or in concert with lysosomes, localize to mitochondria and membrane-bound organelles. Likewise, TFG regulates the abundance of metabolic enzymes, such as ALDOC and the fatty acid-activating enzyme ACOT9. To test consequently for a function of TFG in lipid metabolism, we performed shotgun lipidomics of glycerophospholipids. Total phosphatidylglycerol is more abundant in CH12 tfg KO B cells. Several glycerophospholipid species with similar acyl side chains, such as 36:2 phosphatidylethanolamine and 36:2 phosphatidylinositol, show a dysequilibrium. We suggest a role for TFG in lipid homeostasis, mitochondrial functions, translation, and metabolism in B cells.

Transmissibility and immune evasion of the recently emerged, highly mutated SARS-CoV-2 BA.2.87.1 are unknown. Here, we report that BA.2.87.1 efficiently enters human cells but is more sensitive to antibody-mediated neutralization than the currently dominating JN.1 variant. Acquisition of adaptive mutations might thus be needed for efficient spread in the population.

Glucose uptake increases during B cell activation and antibody-secreting cell (ASC) differentiation, but conflicting findings prevent a clear metabolic profile at different stages of B cell activation. Deletion of the glucose transporter type 1 (GLUT1) gene in mature B cells (GLUT1-cKO) results in normal B cell development, but it reduces germinal center B cells and ASCs. GLUT1-cKO mice show decreased antigen-specific antibody titers after vaccination. In vitro, GLUT1-deficient B cells show impaired activation, whereas established plasmablasts abolish glycolysis, relying on mitochondrial activity and fatty acids. Transcriptomics and metabolomics reveal an altered anaplerotic balance in GLUT1-deficient ASCs. Despite attempts to compensate for glucose deprivation by increasing mitochondrial mass and gene expression associated with glycolysis, the tricarboxylic acid cycle, and hexosamine synthesis, GLUT1-deficient ASCs lack the metabolites for energy production and mitochondrial respiration, limiting protein synthesis. We identify GLUT1 as a critical metabolic player defining the germinal center response and humoral immunity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

BA.2.86, a recently identified descendant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.2 sublineage, contains ∼35 mutations in the spike (S) protein and spreads in multiple countries. Here, we investigated whether the virus exhibits altered biological traits, focusing on S protein-driven viral entry. Employing pseudotyped particles, we show that BA.2.86, unlike other Omicron sublineages, enters Calu-3 lung cells with high efficiency and in a serine- but not cysteine-protease-dependent manner. Robust lung cell infection was confirmed with authentic BA.2.86, but the virus exhibited low specific infectivity. Further, BA.2.86 was highly resistant against all therapeutic antibodies tested, efficiently evading neutralization by antibodies induced by non-adapted vaccines. In contrast, BA.2.86 and the currently circulating EG.5.1 sublineage were appreciably neutralized by antibodies induced by the XBB.1.5-adapted vaccine. Collectively, BA.2.86 has regained a trait characteristic of early SARS-CoV-2 lineages, robust lung cell entry, and evades neutralizing antibodies. However, BA.2.86 exhibits low specific infectivity, which might limit transmissibility., Competing Interests: Declaration of interests A.K., I.N., S.P. and M.H. conducted contract research (testing of vaccinee sera for neutralizing activity against SARS-CoV-2) for Valneva unrelated to this work. G.M.N.B. served as advisor for Moderna, and S.P. served as advisor for BioNTech, unrelated to this work. A.D-J. served as advisor for Pfizer, unrelated to this work. The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: B cell hyperactivity plays an important role in primary Sjögren's syndrome (SS). We undertook this study to better understand the B cell effector branch, namely antibody-secreting cells (ASCs) in primary SS, and to examine the quantity, maturity, and inflammatory properties of ASCs in primary SS patients., Methods: Circulating ASCs, defined as CD3-CD14-CD27+CD38++ cells, from 21 primary SS patients and 10 healthy controls were assessed using spectral flow cytometry. Expression levels of relevant ASC markers relating to maturity, survival, and inflammatory status were analyzed using a t-distributed stochastic neighbor embedding approach. Correlation of ASC properties with primary SS disease parameters was assessed., Results: ASCs were more abundant in peripheral blood from primary SS patients than from healthy controls (mean ± SD 3.1 ± 5.1 cells/μl versus 1.1 ± 1.0 cells/μl, respectively; P = 0.048) and displayed a more mature phenotype (mean ± SD CD19- ASCs 0.37 ± 1.21 cells/μl versus 0.06 ± 0.11 cells/μl, respectively; P = 0.005). An inflammatory CXCR3+ phenotype of ASCs correlated positively with our newly developed ASC maturity index (r = 0.568, P = 0.007) but correlated negatively with antiinflammatory interleukin-10 expression (r = -0.769, P < 0.001). ASCs with a higher maturity index also demonstrated higher levels of the pro-survival protein myeloid cell leukemia 1 (r = 0.567, P = 0.007). Frequency and/or maturity of ASCs correlated with several primary SS disease parameters, such as antinuclear antibody and anti-La/SSB titers, salivary gland focus scores, and ocular staining scores., Conclusion: Quantity and maturity of ASCs in primary SS patients are increased and correlate with disease parameters. A higher maturity index of ASCs marks a pro-survival and proinflammatory phenotype. Altogether, B cell hyperactivity in primary SS extends to the peripheral ASC compartment, raising potential for ASCs as future biomarkers or targets for primary SS treatment., (© 2022 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

We have previously shown that the microRNA (miRNA) processor complex consisting of the RNAse Drosha and the DiGeorge Critical Region (DGCR) 8 protein is essential for B cell maturation. To determine whether miRNA processing is required to initiate T cell-mediated antibody responses, we deleted DGCR8 in maturing B2 cells by crossing a mouse with loxP-flanked DGCR8 alleles with a CD23-Cre mouse. As expected, non-immunized mice showed reduced numbers of mature B2 cells and IgG-secreting cells and diminished serum IgG titers. In accordance, germinal centers and antigen-specific IgG-secreting cells were absent in mice immunized with T-dependent antigens. Therefore, DGCR8 is required to mount an efficient T-dependent antibody response. However, DGCR8 deletion in B1 cells was incomplete, resulting in unaltered B1 cell numbers and normal IgM and IgA titers in DGCR8-knock-out mice. Therefore, this mouse model could be used to analyze B1 responses in the absence of functional B2 cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Daum, Ottmann, Meinzinger, Schulz, Côrte-Real, Hauke, Roth, Schuh, Mielenz, Jäck and Pracht.)

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitates viral entry into host cells and is the key target for neutralizing antibodies. The SARS-CoV-2 lineage B.1.620 carries fifteen mutations in the S protein and is spread in Africa, the US and Europe, while lineage R.1 harbors four mutations in S and infections were observed in several countries, particularly Japan and the US. However, the impact of the mutations in B.1.620 and R.1 S proteins on antibody-mediated neutralization and host cell entry are largely unknown. Here, we report that these mutations are compatible with robust ACE2 binding and entry into cell lines, and they markedly reduce neutralization by vaccine-induced antibodies. Our results reveal evasion of neutralizing antibodies by B.1.620 and R.1, which might have contributed to the spread of these lineages.

To elucidate the function of oxidative phosphorylation (OxPhos) during B cell differentiation, we employ CD23Cre-driven expression of the dominant-negative K320E mutant of the mitochondrial helicase Twinkle (DNT). DNT-expression depletes mitochondrial DNA during B cell maturation, reduces the abundance of respiratory chain protein subunits encoded by mitochondrial DNA, and, consequently, respiratory chain super-complexes in activated B cells. Whereas B cell development in DNT mice is normal, B cell proliferation, germinal centers, class switch to IgG, plasma cell maturation, and T cell-dependent as well as T cell-independent humoral immunity are diminished. DNT expression dampens OxPhos but increases glycolysis in lipopolysaccharide and B cell receptor-activated cells. Lipopolysaccharide-activated DNT-B cells exhibit altered metabolites of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle and a lower amount of phosphatidic acid. Consequently, mTORC1 activity and BLIMP1 induction are curtailed, whereas HIF1α is stabilized. Hence, mitochondrial DNA controls the metabolism of activated B cells via OxPhos to foster humoral immunity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Effective vaccines and monoclonal antibodies have been developed against coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the appearance of virus variants with higher transmissibility and pathogenicity is a major concern because of their potential to escape vaccines and clinically approved SARS-CoV-2- antibodies. Here, we use flow cytometry-based binding and pseudotyped SARS-CoV-2 neutralization assays to determine the efficacy of boost immunization and therapeutic antibodies to neutralize the dominant Omicron variant. We provide compelling evidence that the third vaccination with BNT162b2 increases the amount of neutralizing serum antibodies against Delta and Omicron variants, albeit to a lower degree when compared to the parental Wuhan strain. Therefore, a third vaccination is warranted to increase titers of protective serum antibodies, especially in the case of the Omicron variant. We also found that most clinically approved and otherwise potent therapeutic antibodies against the Delta variant failed to recognize and neutralize the Omicron variant. In contrast, some antibodies under preclinical development potentially neutralized the Omicron variant. Our studies also support using a flow cytometry-based antibody binding assay to rapidly monitor therapeutic candidates and serum titers against emerging SARS-CoV-2 variants., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly isolate human monoclonal antibodies. After immunizing these mice with DNA encoding the spike protein of SARS-CoV-2 and boosting with spike protein, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralize SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of three clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of six clonally related neutralizing antibodies bind to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2-induced weight loss. The two clusters of potent noncompeting SARS-CoV-2 neutralizing antibodies provide potential candidates for therapy and prophylaxis of COVID-19. The study further supports transgenic animals with a human immunoglobulin gene repertoire as a powerful platform in pandemic preparedness initiatives., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Krüppel-like factor 2 (KLF2) is a potent regulator of lymphocyte differentiation, activation and migration. However, its functional role in adaptive and humoral immunity remains elusive. Therefore, by using mice with a B cell-specific deletion of KLF2, we investigated plasma cell differentiation and antibody responses. We revealed that the deletion of KLF2 resulted in perturbed IgA plasma cell compartmentalization, characterized by the absence of IgA plasma cells in the bone marrow, their reductions in the spleen, the blood and the lamina propria of the colon and the small intestine, concomitant with their accumulation and retention in mesenteric lymph nodes and Peyer's patches. Most intriguingly, secretory IgA in the intestinal lumen was almost absent, dimeric serum IgA was drastically reduced and antigen-specific IgA responses to soluble Salmonella flagellin were blunted in KLF2-deficient mice. Perturbance of IgA plasma cell localization was caused by deregulation of CCR9, Integrin chains αM, α4, β7, and sphingosine-1-phosphate receptors. Hence, KLF2 not only orchestrates the localization of IgA plasma cells by fine-tuning chemokine receptors and adhesion molecules but also controls IgA responses to Salmonella flagellin., (© 2022. The Author(s).)

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies., Competing Interests: Declaration of interests All affiliations are listed on the title page of the manuscript. The authors and their immediate family members have no financial interests to declare. The authors have lodged a patent (Australian provisional patent application no. 2021901716) based on this work., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

To monitor infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and successful vaccination against coronavirus disease 2019 (COVID-19), the kinetics of neutralizing or blocking anti-SARS-CoV-2 antibody titers need to be assessed. Here, we report the development of a quick and inexpensive surrogate SARS-CoV-2 blocking assay (SUBA) using immobilized recombinant human angiotensin-converting enzyme 2 (hACE2) and human cells expressing the native form of surface SARS-CoV-2 spike protein. Spike protein-expressing cells bound to hACE2 in the absence or presence of blocking antibodies were quantified by measuring the optical density of cell-associated crystal violet in a spectrophotometer. The advantages are that SUBA is a fast and inexpensive assay, which does not require biosafety level 2- or 3-approved laboratories. Most importantly, SUBA detects blocking antibodies against the native trimeric cell-bound SARS-CoV-2 spike protein and can be rapidly adjusted to quickly pre-screen already approved therapeutic antibodies or sera from vaccinated individuals for their ACE2 blocking activities against any emerging SARS-CoV-2 variants., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Plasma cells depend on quality control of newly synthesized antibodies in the endoplasmic reticulum (ER) via macroautophagy/autophagy and proteasomal degradation. The cytosolic adaptor protein TFG (Trk-fused gene) regulates ER-Golgi transport, the secretory pathway and proteasome activity in non-immune cells. We show here that TFG is upregulated during lipopolysaccharide- and CpG-induced differentiation of B1 and B2 B cells into plasmablasts, with the highest expression of TFG in mature plasma cells. CRISPR-CAS9-mediated gene disruption of tfg in the B lymphoma cell line CH12 revealed increased apoptosis, which was reverted by BCL2 but even more by ectopic TFG expression. Loss of TFG disrupted ER structure, leading to an expanded ER and increased expression of ER stress genes. When compared to wild-type CH12 cells, tfg KO CH12 cells were more sensitive toward ER stress induced by tunicamycin, monensin and proteasome inhibition or by expression of an ER-bound immunoglobulin (Ig) μ heavy (µH) chain. CH12 tfg KO B cells displayed more total LC3, lower LC3-II turnover and increased numbers and size of autophagosomes. Tandem-fluorescent-LC3 revealed less accumulation of GFP-LC3 in starved and chloroquine-treated CH12 tfg KO B cells. The GFP:RFP ratio of tandem-fluorescent-LC3 was higher in tunicamycin-treated CH12 tfg KO B cells, suggesting less autophagy flux during induced ER stress. Based on these data, we suggest that TFG controls autophagy flux in CH12 B cells and propose that TFG is a survival factor that alleviates ER stress through the support of autophagy flux in activated B cells and mature plasma cells. Abbreviations : Ab, antibody; Ag, antigen; ASC, antibody-secreting cells; ATG, autophagy-related; BCR, B cell receptor; COPII, coat protein complex II; CpG, non-methylated CpG oligonucleotide; ER, endoplasmic reticulum; ERAD, ER-associated degradation; FO, follicular; GFP, green fluorescent protein; HC, heavy chain; Ig, immunoglobulin; IRES, internal ribosomal entry site; LC, light chain; MZ, marginal zone; NFKB, nuclear factor of kappa light polypeptide gene enhancer in B cells; TLR, toll-like receptor; UPR, unfolded protein response.

Long-lived antibody-secreting plasma cells are essential to establish humoral memory against pathogens. While a regulatory transcription factor network has been established in plasma cell differentiation, the regulatory role of miRNAs remains enigmatic. We have recently identified miR-148a as the most abundant miRNA in primary mouse and human plasma cells. To determine whether this plasma cell signature miRNA controls the in vivo development of B cells into long-lived plasma cells, we established mice with genomic, conditional, and inducible deletions of miR-148a. The analysis of miR-148a-deficient mice revealed reduced serum Ig, decreased numbers of newly formed plasmablasts and reduced CD19-negative, CD93-positive long-lived plasma cells. Transcriptome and metabolic analysis revealed an impaired glucose uptake, a reduced oxidative phosphorylation-based energy metabolism, and an altered abundance of homing receptors CXCR3 (increase) and CXCR4 (reduction) in miR-148a-deficient plasma cells. These findings support the role of miR-148a as a positive regulator of the maintenance of long-lived plasma cells., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Plasma cells secreting affinity-matured antibodies develop in germinal centers (GCs), where B cells migrate persistently and directionally over defined periods of time. How modes of GC B cell migration influence plasma cell development remained unclear. Through genetic deletion of the F-actin bundling protein Swiprosin-1/EF-hand domain family member 2 (EFhd2) and by two-photon microscopy, we show that EFhd2 restrains B cell speed in GCs and hapten-specific plasma cell output. Modeling the GC reaction reveals that increasing GC B cell speed promotes plasma cell generation. Lack of EFhd2 also reduces contacts of GC B cells with follicular dendritic cells in vivo. Computational modeling uncovers that both GC output and antibody affinity depend quantitatively on contacts of GC B cells with follicular dendritic cells when B cells migrate more persistently. Collectively, our data explain how GC B cells integrate speed and persistence of cell migration with B cell receptor affinity., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The sensing of microbial genetic material by leukocytes often elicits beneficial pro-inflammatory cytokines, but dysregulated responses can cause severe pathogenesis. Genome-wide association studies have linked the gene encoding phospholipase D3 (PLD3) to Alzheimer's disease and have linked PLD4 to rheumatoid arthritis and systemic sclerosis. PLD3 and PLD4 are endolysosomal proteins whose functions are obscure. Here, PLD4-deficient mice were found to have an inflammatory disease, marked by elevated levels of interferon-γ (IFN-γ) and splenomegaly. These phenotypes were traced to altered responsiveness of PLD4-deficient dendritic cells to ligands of the single-stranded DNA sensor TLR9. Macrophages from PLD3-deficient mice also had exaggerated TLR9 responses. Although PLD4 and PLD3 were presumed to be phospholipases, we found that they are 5' exonucleases, probably identical to spleen phosphodiesterase, that break down TLR9 ligands. Mice deficient in both PLD3 and PLD4 developed lethal liver inflammation in early life, which indicates that both enzymes are needed to regulate inflammatory cytokine responses via the degradation of nucleic acids.

We provide a robust four-color fluorescence-based flow cytometry protocol that distinguishes viable dividing plasmablasts from nondividing plasma cells and, based on CD19 surface abundance, identifies two mature plasma cell populations in the spleen and the bone marrow of mice., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

microRNAs (miRNAs) are important posttranscriptional regulators during hematopoietic lineage commitment and lymphocyte development. Mature miRNAs are processed from primary miRNA transcripts in two steps by the microprocessor complex, consisting of Drosha and its partner DiGeorge Critical Region 8 (DGCR8), and the RNAse III enzyme, Dicer. Conditional ablations of Drosha and Dicer have established the importance of both RNAses in B- and T-cell development. Here, we show that a cre-mediated B-cell specific deletion of DGCR8 in mice results in a nearly complete maturation block at the transition from the pro-B to the pre-B cell stage, and a failure to upregulate Ig μ heavy chain expression in pro-B cells. Furthermore, we found that the death of freshly isolated DGCR8-deficient pro-B cells could be partially prevented by enforced Bcl2 expression. We conclude from these findings that the microprocessor component DGCR8 is essential for survival and differentiation of early B-cell progenitors., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Population-based studies on Staphylococcus aureus nasal colonization are scarce. We examined the prevalence, resistance, and molecular diversity of S. aureus in the general population in Northeast Germany. Nasal swabs were obtained from 3,891 adults in the large-scale population-based Study of Health in Pomerania (SHIP-TREND). Isolates were characterized using spa genotyping, as well as antibiotic resistance and virulence gene profiling. We observed an S. aureus prevalence of 27.2%. Nasal S. aureus carriage was associated with male sex and inversely correlated with age. Methicillin-resistant S. aureus (MRSA) accounted for 0.95% of the colonizing S. aureus strains. MRSA carriage was associated with frequent visits to hospitals, nursing homes, or retirement homes within the previous 24 months. All MRSA strains were resistant to multiple antibiotics. Most MRSA isolates belonged to the pandemic European hospital-acquired MRSA sequence type 22 (HA-MRSA-ST22) lineage. We also detected one livestock-associated MRSA ST398 (LA-MRSA-ST398) isolate, as well as six livestock-associated methicillin-susceptible S. aureus (LA-MSSA) isolates (clonal complex 1 [CC1], CC97, and CC398). spa typing revealed a diverse but also highly clonal S. aureus population structure. We identified a total of 357 spa types, which were grouped into 30 CCs or sequence types. The major seven CCs (CC30, CC45, CC15, CC8, CC7, CC22, and CC25) included 75% of all isolates. Virulence gene patterns were strongly linked to the clonal background. In conclusion, MSSA and MRSA prevalences and the molecular diversity of S. aureus in Northeast Germany are consistent with those of other European countries. The detection of HA-MRSA and LA-MRSA within the general population indicates possible transmission from hospitals and livestock, respectively, and should be closely monitored., (Copyright © 2016 Holtfreter et al.)

Friedreich ataxia (FRDA) is a progressive neurodegenerative disorder associated with ataxia, dysarthria, pyramidal tract signs, sensory loss, cardiomyopathy and diabetes. There is no cure for FRDA so far. Studies of the natural history of the disease and future therapeutic trials require development of appropriate outcome markers. Since any therapeutic benefit is expected to modulate deterioration over time rather than to reverse disability, potential outcome measures must be sensitive instruments carefully analysed for their significance. Clinical scales may represent an appropriate measuring tool. Over the last few years the construction, evaluation and validation of sensitive clinical scales for the assessment of disease severity and progression in ataxia have had considerable impact on our understanding of the disease. Currently, there are three different scales that are most frequently applied: The International Cooperative Ataxia Rating Scale (ICARS), the Friedreich Ataxia Rating Scale (FARS) and the Scale for the Assessment and Rating of Ataxia (SARA). All scales have been validated and compared with regard to their testing properties., (© 2013 International Society for Neurochemistry.)

A series of symmetrical and unsymmetrical N,N'-disubstituted aminotroponimines (ATIHs) have been prepared. Substituents ranging from linear to cyclic alkyl groups, chelating ethers, and aryl groups were employed. The corresponding aminotroponiminate zinc complexes were then synthesized and characterized by a number of techniques, including by X-ray crystallography. Herein we report on the investigations into their activity in the intramolecular hydroamination of nonactivated alkenes. We also demonstrate that complexes bearing ligands with cyclic alkyl groups show superior activity in a number of selected reactions with functionalized aminoalkenes.

The long-term effects of high bone resorption on blood ionized calcium and calciotropic hormone levels following oophorectomy in 6-month-old Sprague-Dawley female rats were investigated. Fasting urine and blood samples were collected from 16 sham and 16 oophorectomized (oophx) rats preoperatively and up to 130 days postoperatively. From 50 days postoperation, daily injections of 17-beta estradiol (E2) (20 microg/kg body weight) were administered subcutaneously to eight of the oophx rats. Urine hydroxyproline excretion (OHPrE) and serum osteocalcin were significantly elevated (P < 0.001) as a result of oophorectomy and normalized within 6 days of E2 replacement. Urine deoxypyridinoline and total serum alkaline phosphatase were significantly elevated (P < 0.001) following oophorectomy and suppressed to control levels after 37 days of E2 replacement. Blood ionized calcium was significantly reduced in oophx rats (P < 0.001) compared with sham rats and was normalized by E2 replacement at 55 days posttreatment. Serum 1,25 dihydroxyvitamin D (1,25(OHD)2D3) was significantly elevated (P < 0.001) in oophx rats and again was normalized by E2 at 55 days posttreatment. Serum parathyroid hormone (PTH) was unaffected by oophorectomy. These data indicate that despite increased bone resorption following oophorectomy, blood ionized calcium levels are decreased. The increased bone turnover in oophx rats was rapidly suppressed by E2 replacement before ionized calcium levels were normalized, suggesting a direct effect of estrogen on the modulation of bone cell activity. The depression of blood ionized calcium levels following oophorectomy, which is not mediated by calciotropic hormones, suggests an effect of estrogen on intestinal calcium absorption, renal handling of calcium, or a combination of both.

Postmenopausal bone loss is associated with a rise in the fasting urine calcium excretion which has been proposed to be a possible cause of increased bone resorption. The oophorectomized rat is widely used as a model of postmenopausal bone loss, but preliminary data suggest that urine calcium excretion is not increased following oophorectomy in this animal model. However the present detailed experimental protocol demonstrated that oophorectomy in adult rats increased the obligatory urine calcium excretion compared with ovary-intact rats (P < 0.001). Importantly urine hydroxyproline excretion was positively correlated with urine calcium only in the oophorectomized rats (P = 0.003). This urine calcium was positively correlated with urine phosphate excretion when dietary sodium was low (P < 0.001) and with urine sodium excretion when dietary sodium was elevated (P = 0.003). Calcium balance studies indicated a greater fall in calcium accretion in growing, oophorectomized rats (6 to 15 weeks of age) compared with ovary-intact animals and intestinal calcium secretion was the major component of the calcium balance to be affected. Losses of calcium in the urine were relatively minor in these growing rats.