MIWI N-terminal arginines orchestrate generation of functional pachytene piRNAs and spermiogenesis.

N-terminal arginine (NTR) methylation is a conserved feature of PIWI proteins, which are central components of the PIWI-interacting RNA (piRNA) pathway. The significance and precise function of PIWI NTR methylation in mammals remains unknown. In mice, PIWI NTRs bind Tudor domain containing proteins (TDRDs) that have essential roles in piRNA biogenesis and the formation of the chromatoid body. Using mouse MIWI (PIWIL1) as paradigm, we demonstrate that the NTRs are essential for spermatogenesis through the regulation of transposons and gene expression. The loss of TDRD5 and TDRKH interaction with MIWI results in attenuation of piRNA amplification. We find that piRNA amplification is necessary for transposon control and for sustaining piRNA levels including select, nonconserved, pachytene piRNAs that target specific mRNAs required for spermatogenesis. Our findings support the notion that the vast majority of pachytene piRNAs are dispensable, acting as self-serving genetic elements that rely for propagation on MIWI piRNA amplification. MIWI-NTRs also mediate interactions with TDRD6 that are necessary for chromatoid body compaction. Furthermore, MIWI-NTRs promote stabilization of spermiogenic transcripts that drive nuclear compaction, which is essential for sperm formation. In summary, the NTRs underpin the diversification of MIWI protein function.

Diversity of major histocompatibility complex (MHC) and natural killer cell receptor (NKR) genes and their interactions in domestic horses.

The immunogenome is the part of the genome that underlies immune mechanisms and evolves under various selective pressures. Two complex regions of the immunogenome, major histocompatibility complex (MHC) and natural killer cell receptor (NKR) genes, play an important role in the response to selective pressures of pathogens. Their importance is expressed by their genetic polymorphism at the molecular level, and their diversity associated with different types of diseases at the population level. Findings of associations between specific combinations of MHC/NKR haplotypes with different diseases in model species suggest that these gene complexes did not evolve independently. No such associations have been described in horses so far. The aim of the study was to detect associations between MHC and NKR gene/microsatellite haplotypes in three horse breed groups (Camargue, African, and Romanian) by statistical methods; chi-square test, Fisher's exact test, Pearson's goodness-of-fit test and logistic regression. Associations were detected for both MHC/NKR genes and microsatellites; the most significant associations were found between the most variable KLRA3 gene and the EQCA-1 or EQCA-2 genes. This finding supports the assumption that the KLRA3 is an important receptor for MHC I and that interactions of these molecules play important roles in the horse immunity and reproduction. Despite some limitations of the study such as low numbers of horses or lack of knowledge of the selected genes functions, the results were consistent across different statistical methods and remained significant even after overconservative Bonferroni corrections. We therefore consider them biologically plausible.

MIWI arginines orchestrate generation of functional pachytene piRNAs and spermiogenesis.

N-terminal arginine (NTR) methylation is a conserved feature of PIWI proteins, which are central components of the PIWI-interacting RNA (piRNA) pathway. The significance and precise function of PIWI NTR methylation in mammals remains unknown. In mice, PIWI NTRs bind Tudor domain containing proteins (TDRDs) that have essential roles in piRNA biogenesis and the formation of the chromatoid body. Using mouse MIWI (PIWIL1) as paradigm, we demonstrate that the NTRs are essential for spermatogenesis through the regulation of transposons and gene expression. Surprisingly, the loss of TDRD5 and TDRKH interaction with MIWI results in defective piRNA amplification, rather than an expected failure of piRNA biogenesis. We find that piRNA amplification is necessary for both transposon control and for sustaining levels of select, nonconserved, pachytene piRNAs that target specific mRNAs required for spermatogenesis. Our findings support the notion that the vast majority of pachytene piRNAs are dispensable, acting as autonomous genetic elements that rely for propagation on MIWI piRNA amplification. MIWI-NTRs also mediate interactions with TDRD6 that are necessary for chromatoid body compaction. Furthermore, MIWI-NTRs promote stabilization of spermiogenic transcripts that drive nuclear compaction, which is essential for sperm formation. In summary, the NTRs underpin the diversification of MIWI protein function.

Low genetic diversity of Treponema pallidum ssp. pertenue (TPE) isolated from patients' ulcers in Namatanai District of Papua New Guinea: Local human population is infected by three TPE genotypes.

Yaws is an endemic disease caused by Treponema pallidum subsp. pertenue (TPE) that primarily affects children in rural regions of the tropics. The endemic character of yaws infections and the expected exclusive reservoir of TPE in humans opened a new opportunity to start a yaws eradication campaign. We have developed a multi-locus sequence typing (MLST) scheme for TPE isolates combining the previously published (TP0548, TP0488) and new (TP0858) chromosomal loci, and we compared this typing scheme to the two previously published MLST schemes. We applied this scheme to TPE-containing clinical isolates obtained during a mass drug administration study performed in the Namatanai District of Papua New Guinea between June 2018 and December 2019. Of 1081 samples collected, 302 (28.5%) tested positive for TPE DNA, from which 255 (84.4%) were fully typed. The TPE PCR-positivity in swab samples was higher in younger patients, patients with single ulcers, first ulcer episodes, and with ulcer duration less than six months. Non-treponemal serological test positivity correlated better with PCR positivity compared to treponema-specific serological tests. The MLST revealed a low level of genetic diversity among infecting TPE isolates, represented by just three distinct genotypes (JE11, SE22, and TE13). Two previously used typing schemes revealed similar typing resolutions. Two new alleles (one in TP0858 and one in TP0136) were shown to arise by intragenomic recombination/deletion events. Compared to samples genotyped as JE11, the minor genotypes (TE13 and SE22) were more frequently detected in samples from patients with two or more ulcers and patients with higher values of specific TP serological tests. Moreover, the A2058G mutation in the 23S rRNA genes of three JE11 isolates was found, resulting in azithromycin resistance.

Light-responsive microRNA molecules in human retinal organoids are differentially regulated by distinct wavelengths of light.

Cells in the human retina must rapidly adapt to constantly changing visual stimuli. This fast adaptation to varying levels and wavelengths of light helps to regulate circadian rhythms and allows for adaptation to high levels of illumination, thereby enabling the rest of the visual system to remain responsive. It has been shown that retinal microRNA (miRNA) molecules play a key role in regulating these processes. However, despite extensive research using various model organisms, light-regulated miRNAs in human retinal cells remain unknown. Here, we aim to characterize these miRNAs. We generated light-responsive human retinal organoids that express miRNA families and clusters typically found in the retina. Using an in-house developed photostimulation device, we identified a subset of light-regulated miRNAs. Importantly, we found that these miRNAs are differentially regulated by distinct wavelengths of light and have a rapid turnover, highlighting the dynamic and adaptive nature of the human retina.

Newly identified variability of the antigen binding site coding sequences of the equine major histocompatibility complex class I and class II genes.

The major histocompatibility complex (MHC) with its class I and II genes plays a crucial role in the immune response to pathogens by presenting oligopeptide antigens to various immune response effector cells. In order to counteract the vast variability of infectious agents, MHC class I and II genes usually retain high levels of SNPs mainly concentrated in the exons encoding the antigen binding sites. The aim of the study was to reveal new variability of selected MHC genes with a special focus on MHC class I physical haplotypes. Long-range NGS to was used to identify exon 2-exon 3 alleles in three genetically distinct horse breeds. A total of 116 allelic variants were found in the MHC class I genes Eqca-1, Eqca-2, Eqca-7 and Eqca-Ψ, 112 of which were novel. The MHC class II DRA locus was confirmed to comprise five exon 2 alleles, and no new sequences were observed. Additional variability in terms of 15 novel exon 2 alleles was identified in the DQA1 locus. Extensive overall variability across the entire MHC region was confirmed by an analysis of MHC-linked microsatellite loci. Both diversifying and purifying selection were detected within the MHC class I and II loci analyzed.

MicroRNA Profiling of Self-Renewing Human Neural Stem Cells Reveals Novel Sets of Differentially Expressed microRNAs During Neural Differentiation In Vitro.

The involvement of microRNAs (miRNAs) in orchestrating self-renewal and differentiation of stem cells has been revealed in a number of recent studies. And while in human pluripotent stem cells, miRNAs have been directly linked to the core pluripotency network, including the cell cycle regulation and the maintenance of the self-renewing capacity, their role in the onset of differentiation in other contexts, such as determination of neural cell fate, remains poorly described. To bridge this gap, we used three model cell types to study miRNA expression patterns: human embryonic stem cells (hESCs), hESCs-derived self-renewing neural stem cells (NSCs), and differentiating NSCs. The comprehensive miRNA profiling presented here reveals novel sets of miRNAs differentially expressed during human neural cell fate determination in vitro. Furthermore, we report a miRNA expression profile of self-renewing human NSCs, which has been lacking to this date. Our data also indicates that miRNA clusters enriched in NSCs share the target-determining seed sequence with cell cycle regulatory miRNAs expressed in pluripotent hESCs. Lastly, our mechanistic experiments confirmed that cluster miR-17-92, one of the NSCs-enriched clusters, is directly transcriptionally regulated by transcription factor c-MYC.

The natural cytotoxicity receptor genes in the family Felidae.

Natural killer (NK) cells belong to the innate immune system. The germline-encoded natural killer cell receptors represent activating and inhibitory receptors regulating multiple NK cell activities. The natural cytotoxicity receptors (NCRs) are activating natural cytotoxicity triggering receptors 1, 2, and 3 (NKp46, NKp44, and NKp30), encoded by the genes NCR1, NCR2, and NCR3, respectively. NCRs may be expressed in different cell types engaged in mechanisms of innate and adaptive immunity. The family Felidae, comprising the domestic cat and a wide variety of free-ranging species represents a well-suited model for biomedical and evolutionary studies. We characterized the NCR1, NCR2, and NCR3 genes in a panel of felid species. We confirmed the presence of potentially functional genes NCR1, NCR2, and NCR3 in all species. All three genes are conserved within the family and are similar to other phylogenetically related mammalian families. The NCR1 and NCR2 phylogenetic trees based on both nucleotide and protein sequences corresponded to the current zoological taxonomy, with some exceptions suggesting effects of different selection pressures in some species. Highly conserved NCR3 sequences did not allow a robust phylogenetic analysis. Most interspecific differences both at the nucleotide and protein level were found in NCR2. Within species, the most polymorphic CDS was detected in NCR1. Selection analyses indicated the effects of purifying selection on individual amino acid sites in all three genes. In stray cats, a rather high intraspecific diversity was observed.

Whole genome sequences of Treponema pallidum subsp. endemicum isolated from Cuban patients: The non-clonal character of isolates suggests a persistent human infection rather than a single outbreak.

Bejel (endemic syphilis) is a neglected non-venereal disease caused by Treponema pallidum subsp. endemicum (TEN). Although it is mostly present in hot, dry climates, a few cases have been found outside of these areas. The aim of this work was the sequencing and analysis of TEN isolates obtained from "syphilis patients" in Cuba, which is not considered an endemic area for bejel. Genomes were obtained by pool segment genome sequencing or direct sequencing methods, and the bioinformatics analysis was performed according to an established pipeline. We obtained four genomes with 100%, 81.7%, 52.6%, and 21.1% breadth of coverage, respectively. The sequenced genomes revealed a non-clonal character, with nucleotide variability ranging between 0.2-10.3 nucleotide substitutions per 100 kbp among the TEN isolates. Nucleotide changes affected 27 genes, and the analysis of the completely sequenced genome also showed a recombination event between tprC and tprI, in TP0488 as well as in the intergenic region between TP0127-TP0129. Despite limitations in the quality of samples affecting breadth of sequencing coverage, the determined non-clonal character of the isolates suggests a persistent infection in the Cuban population rather than a single outbreak caused by imported case.

KMT2C methyltransferase domain regulated INK4A expression suppresses prostate cancer metastasis.

BACKGROUND: Frequent truncation mutations of the histone lysine N-methyltransferase KMT2C have been detected by whole exome sequencing studies in various cancers, including malignancies of the prostate. However, the biological consequences of these alterations in prostate cancer have not yet been elucidated. METHODS: To investigate the functional effects of these mutations, we deleted the C-terminal catalytic core motif of Kmt2c specifically in mouse prostate epithelium. We analysed the effect of Kmt2c SET domain deletion in a Pten-deficient PCa mouse model in vivo and of truncation mutations of KMT2C in a large number of prostate cancer patients. RESULTS: We show here for the first time that impaired KMT2C methyltransferase activity drives proliferation and PIN formation and, when combined with loss of the tumour suppressor PTEN, triggers loss of senescence, metastatic dissemination and dramatically reduces life expectancy. In Kmt2c-mutated tumours we show enrichment of proliferative MYC gene signatures and loss of expression of the cell cycle repressor p16INK4A. In addition, we observe a striking reduction in disease-free survival of patients with KMT2C-mutated prostate cancer. CONCLUSIONS: We identified truncating events of KMT2C as drivers of proliferation and PIN formation. Loss of PTEN and KMT2C in prostate cancer results in loss of senescence, metastatic dissemination and reduced life expectancy. Our data demonstrate the prognostic significance of KMT2C mutation status in prostate cancer patients. Inhibition of the MYC signalling axis may be a viable treatment option for patients with KMT2C truncations and therefore poor prognosis.

The deadly face of felid killer cells: The cytotoxic proteins and their genes.

Natural killer cells and cytotoxic T lymphocytes are the main cell populations of the immune system able to directly kill target cells via cytotoxic granules. Different mammalian species may differ in specific features of their pore-forming protein (perforin) and granule-bound serine proteases (granzymes). One perforin gene (PRF1) and four genes encoding granzymes A, B, H, and K (GZMA, GZMB, GZMH, GZMK) were identified in the reference genomes of felids. The objective of this work was to characterize the genes PRF1, GZMA and GZMB in a panel of 17 felid species by next-generation re-sequencing. A search of available felid genomes (17 species) retrieved the coding sequences of these genes for comparison to our data. Both sets of sequences or their combinations (23 species) were used for phylogenetic and selection analyses. Nucleotide PRF1, GZMA and GZMB sequences showed high similarities between felid species (over 95% identity). All trees derived from coding sequences expressed phylogenetic relationships corresponding to the zoological taxonomy of the Felidae, except GZMA. No effects of positive selection were detected in the genes studied, however, effects of purifying selection were observed for PRF1 and GZMA. The conservation of PRF1 is in agreement with its critical biological function. The differentiation observed between granzyme sub-families may reflect an adaptation to pathogen variation. The need to maintain important gene functions and at the same time cope with various pathogens may lead to an equilibrium between positive and negative selective pressures acting on GZMB. The within-species variability in wild felid populations merits further investigation.

Genetic diversity of Leptospira isolates in Lao PDR and genome analysis of an outbreak strain.

BACKGROUND: Although Southeast Asia is one of the most leptospirosis afflicted regions, little is known about the diversity and molecular epidemiology of the causative agents of this widespread and emerging zoonotic disease. METHODOLOGY/PRINCIPAL FINDINGS: We used whole genome sequencing to examine genetic variation in 75 Leptospira strains isolated from patients in the Lao PDR (Laos) between 2006 and 2017. Eleven serogroups from 4 Leptospira species and 43 cgMLST-defined clonal groups (CGs) were identified. The most prevalent CG was CG272 (n = 18, 26.8%), composed of L. interrogans serogroup Autumnalis isolates. This genotype was recovered throughout the 12-year period and was associated with deaths, and with a large outbreak in neighbouring Thailand. Genome analysis reveals that the CG272 strains form a highly clonal group of strains that have, for yet unknown reasons, recently spread in Laos and Thailand. Additionally, accessory genes clearly discriminate CG272 strains from the other Leptospira strains. CONCLUSIONS/SIGNIFICANCE: The present study reveals a high diversity of Leptospira genotypes in Laos, thus extending our current knowledge of the pan- and core-genomes of these life-threatening pathogens. Our results demonstrate that the CG272 strains belong to a unique clonal group, which probably evolved through clonal expansion following niche adaptation. Additional epidemiological studies are required to better evaluate the spread of this genotype in Southeast Asia. To further investigate the key factors driving the virulence and spread of these pathogens, more intense genomic surveillance is needed, combining detailed clinical and epidemiological data.

Small RNA Sequencing Identifies PIWI-Interacting RNAs Deregulated in Glioblastoma-piR-9491 and piR-12488 Reduce Tumor Cell Colonies In Vitro.

Glioblastoma (GBM) is the most frequently occurring primary malignant brain tumor of astrocytic origin. To change poor prognosis, it is necessary to deeply understand the molecular mechanisms of gliomagenesis and identify new potential biomarkers and therapeutic targets. PIWI-interacting RNAs (piRNAs) help in maintaining genome stability, and their deregulation has already been observed in many tumors. Recent studies suggest that these molecules could also play an important role in the glioma biology. To determine GBM-associated piRNAs, we performed small RNA sequencing analysis in the discovery set of 19 GBM and 11 non-tumor brain samples followed by TaqMan qRT-PCR analyses in the independent set of 77 GBM and 23 non-tumor patients. Obtained data were subsequently bioinformatically analyzed. Small RNA sequencing revealed 58 significantly deregulated piRNA molecules in GBM samples in comparison with non-tumor brain tissues. Deregulation of piR-1849, piR-9491, piR-12487, and piR-12488 was successfully confirmed in the independent groups of patients and controls (all p < 0.0001), and piR-9491 and piR-12488 reduced GBM cells' ability to form colonies in vitro. In addition, piR-23231 was significantly associated with the overall survival of the GBM patients treated with Stupp regimen (p = 0.007). Our results suggest that piRNAs could be a novel promising diagnostic and prognostic biomarker in GBM potentially playing important roles in gliomagenesis.

Super-enhancer-based identification of a BATF3/IL-2R-module reveals vulnerabilities in anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL), an aggressive CD30-positive T-cell lymphoma, comprises systemic anaplastic lymphoma kinase (ALK)-positive, and ALK-negative, primary cutaneous and breast implant-associated ALCL. Prognosis of some ALCL subgroups is still unsatisfactory, and already in second line effective treatment options are lacking. To identify genes defining ALCL cell state and dependencies, we here characterize super-enhancer regions by genome-wide H3K27ac ChIP-seq. In addition to known ALCL key regulators, the AP-1-member BATF3 and IL-2 receptor (IL2R)-components are among the top hits. Specific and high-level IL2R expression in ALCL correlates with BATF3 expression. Confirming a regulatory link, IL-2R-expression decreases following BATF3 knockout, and BATF3 is recruited to IL2R regulatory regions. Functionally, IL-2, IL-15 and Neo-2/15, a hyper-stable IL-2/IL-15 mimic, accelerate ALCL growth and activate STAT1, STAT5 and ERK1/2. In line, strong IL-2Rα-expression in ALCL patients is linked to more aggressive clinical presentation. Finally, an IL-2Rα-targeting antibody-drug conjugate efficiently kills ALCL cells in vitro and in vivo. Our results highlight the importance of the BATF3/IL-2R-module for ALCL biology and identify IL-2Rα-targeting as a promising treatment strategy for ALCL.

TERA-Seq: true end-to-end sequencing of native RNA molecules for transcriptome characterization.

Direct sequencing of single, native RNA molecules through nanopores has a strong potential to transform research in all aspects of RNA biology and clinical diagnostics. The existing platform from Oxford Nanopore Technologies is unable to sequence the very 5' ends of RNAs and is limited to polyadenylated molecules. Here, we develop True End-to-end RNA Sequencing (TERA-Seq), a platform that addresses these limitations, permitting more thorough transcriptome characterization. TERA-Seq describes both poly- and non-polyadenylated RNA molecules and accurately identifies their native 5' and 3' ends by ligating uniquely designed adapters that are sequenced along with the transcript. We find that capped, full-length mRNAs in human cells show marked variation of poly(A) tail lengths at the single molecule level. We report prevalent capping downstream of canonical transcriptional start sites in otherwise fully spliced and polyadenylated molecules. We reveal RNA processing and decay at single molecule level and find that mRNAs decay cotranslationally, often from their 5' ends, while frequently retaining poly(A) tails. TERA-Seq will prove useful in many applications where true end-to-end direct sequencing of single, native RNA molecules and their isoforms is desirable.

The Population Diversity of Candidate Genes for Resistance/Susceptibility to Coronavirus Infection in Domestic Cats: An Inter-Breed Comparison.

Feline coronavirus (FCoV) is a complex pathogen causing feline infectious peritonitis (FIP). Host genetics represents a factor contributing to the pathogenesis of the disease. Differential susceptibility of various breeds to FIP was reported with controversial results. The objective of this study was to compare the genetic diversity of different breeds on a panel of candidate genes potentially affecting FCoV infection. One hundred thirteen cats of six breeds were genotyped on a panel of sixteen candidate genes. SNP allelic/haplotype frequencies were calculated; pairwise FST and molecular variance analyses were performed. Principal coordinate (PCoA) and STRUCTURE analyses were used to infer population structure. Interbreed differences in allele frequencies were observed. PCoA analysis performed for all genes of the panel indicated no population substructure. In contrast to the full marker set, PCoA of SNP markers associated with FCoV shedding (NCR1 and SLX4IP) showed three clusters containing only alleles associated with susceptibility to FCoV shedding, homozygotes and heterozygotes for the susceptibility alleles, and all three genotypes, respectively. Each cluster contained cats of multiple breeds. Three clusters of haplotypes were identified by PCoA, two clusters by STRUCTURE. Haplotypes of a single gene (SNX5) differed significantly between the PCoA clusters.

Dynamic miRNA changes during the process of epileptogenesis in an infantile and adult-onset model.

Temporal lobe epilepsy (TLE) is the most common epilepsy type. TLE onset in infancy aggravates features like severity, drug responsiveness, or development of comorbidities. These aggravations may arise from altered micro RNA (miRNA) expression specific to the early onset of the disease. Although the miRNA involvement in TLE is widely studied, the relationship between the onset-age and miRNA expression has not been addressed. Here, we investigated the miRNA profile of infantile and adult-onset TLE in rats combining sequencing and PCR. Since miRNA expression changes with the disease progression, we scrutinized miRNA dynamics across three stages: acute, latent, and chronic. We report that infantile-onset TLE leads to changes in the expression of fewer miRNAs across these stages. Interestingly, the miRNA profile in the acute stage of infantile-onset TLE overlaps in dysregulation of miR-132-5p, -205, and -211-3p with the chronic stage of the disease starting in adulthood. The analysis of putative targets linked the majority of dysregulated miRNAs with pathways involved in epilepsy. Our profiling uncovered miRNA expression characteristic for infantile and adulthood-onset epileptogenesis, suggesting the distinct biology underlying TLE in the onset age-dependent matter. Our results indicate the necessity of addressing the onset age as an important parameter in future epilepsy research.

Selective Activation of CNS and Reference PPARGC1A Promoters Is Associated with Distinct Gene Programs Relevant for Neurodegenerative Diseases.

The transcriptional regulator peroxisome proliferator activated receptor gamma coactivator 1A (PGC-1α), encoded by PPARGC1A, has been linked to neurodegenerative diseases. Recently discovered CNS-specific PPARGC1A transcripts are initiated far upstream of the reference promoter, spliced to exon 2 of the reference gene, and are more abundant than reference gene transcripts in post-mortem human brain samples. The proteins translated from the CNS and reference transcripts differ only at their N-terminal regions. To dissect functional differences between CNS-specific isoforms and reference proteins, we used clustered regularly interspaced short palindromic repeats transcriptional activation (CRISPRa) for selective endogenous activation of the CNS or the reference promoters in SH-SY5Y cells. Expression and/or exon usage of the targets was ascertained by RNA sequencing. Compared to controls, more differentially expressed genes were observed after activation of the CNS than the reference gene promoter, while the magnitude of alternative exon usage was comparable between activation of the two promoters. Promoter-selective associations were observed with canonical signaling pathways, mitochondrial and nervous system functions and neurological diseases. The distinct N-terminal as well as the shared downstream regions of PGC-1α isoforms affect the exon usage of numerous genes. Furthermore, associations of risk genes of amyotrophic lateral sclerosis and Parkinson's disease were noted with differentially expressed genes resulting from the activation of the CNS and reference gene promoter, respectively. Thus, CNS-specific isoforms markedly amplify the biological functions of PPARGC1A and CNS-specific isoforms and reference proteins have common, complementary and selective functions relevant for neurodegenerative diseases.

FoxO1-GAB1 axis regulates homing capacity and tonic AKT activity in chronic lymphocytic leukemia.

Recirculation of chronic lymphocytic leukemia (CLL) cells between the peripheral blood and lymphoid niches plays a critical role in disease pathophysiology, and inhibiting this process is one of the major mechanisms of action for B-cell receptor (BCR) inhibitors such as ibrutinib and idelalisib. Migration is a complex process guided by chemokine receptors and integrins. However, it remains largely unknown how CLL cells integrate multiple migratory signals while balancing survival in the peripheral blood and the decision to return to immune niches. Our study provided evidence that CXCR4/CD5 intraclonal subpopulations can be used to study the regulation of migration of CLL cells. We performed RNA profiling of CXCR4dimCD5bright vs CXCR4brightCD5dim CLL cells and identified differential expression of dozens of molecules with a putative function in cell migration. GRB2-associated binding protein 1 (GAB1) positively regulated CLL cell homing capacity of CXCR4brightCD5dim cells. Gradual GAB1 accumulation in CLL cells outside immune niches was mediated by FoxO1-induced transcriptional GAB1 activation. Upregulation of GAB1 also played an important role in maintaining basal phosphatidylinositol 3-kinase (PI3K) activity and the "tonic" AKT phosphorylation required to sustain the survival of resting CLL B cells. This finding is important during ibrutinib therapy, because CLL cells induce the FoxO1-GAB1-pAKT axis, which represents an adaptation mechanism to the inability to home to immune niches. We have demonstrated that GAB1 can be targeted therapeutically by novel GAB1 inhibitors, alone or in combination with BTK inhibition. GAB1 inhibitors induce CLL cell apoptosis, impair cell migration, inhibit tonic or BCR-induced AKT phosphorylation, and block compensatory AKT activity during ibrutinib therapy.

A Deadly Cargo: Gene Repertoire of Cytotoxic Effector Proteins in the Camelidae.

Cytotoxic T cells and natural killer cells can kill target cells based on their expression and release of perforin, granulysin, and granzymes. Genes encoding these molecules have been only poorly annotated in camelids. Based on bioinformatic analyses of genomic resources, sequences corresponding to perforin, granulysin, and granzymes were identified in genomes of camelids and related ungulate species, and annotation of the corresponding genes was performed. A phylogenetic tree was constructed to study evolutionary relationships between the species analyzed. Re-sequencing of all genes in a panel of 10 dromedaries and 10 domestic Bactrian camels allowed analyzing their individual genetic polymorphisms. The data showed that all extant Old World camelids possess functional genes for two pore-forming proteins (PRF1, GNLY) and six granzymes (GZMA, GZMB, GZMH, GZMK, GZMM, and GZMO). All these genes were represented as single copies in the genome except the GZMH gene exhibiting interspecific differences in the number of loci. High protein sequence similarities with other camelid and ungulate species were observed for GZMK and GZMM. The protein variability in dromedaries and Bactrian camels was rather low, except for GNLY and chymotrypsin-like granzymes (GZMB, GZMH).