Mycoplasma DnaK expression increases cancer development in vivo upon DNA damage.

Well-controlled repair mechanisms are involved in the maintenance of genomic stability, and their failure can precipitate DNA abnormalities and elevate tumor risk. In addition, the tumor microenvironment, enriched with factors inducing oxidative stress and affecting cell cycle checkpoints, intensifies DNA damage when repair pathways falter. Recent research has unveiled associations between certain bacteria, including Mycoplasmas, and various cancers, and the causative mechanism(s) are under active investigation. We previously showed that Mycoplasma fermentans DnaK, an HSP70 family chaperone protein, hampers the activity of proteins like PARP1 and p53, crucial for genomic integrity. Moreover, our analysis of its interactome in human cancer cell lines revealed DnaK's engagement with several components of DNA-repair machinery. Finally, in vivo experiments performed in our laboratory using a DnaK knock-in mouse model generated by our group demonstrated that DnaK exposure led to increased DNA copy number variants, indicative of genomic instability. We present here evidence that expression of DnaK is linked to increased i) incidence of tumors in vivo upon exposure to urethane, a DNA damaging agent; ii) spontaneous DNA damage ex vivo; and iii) expression of proinflammatory cytokines ex vivo, variations in reactive oxygen species levels, and increased ß-galactosidase activity across tissues. Moreover, DnaK was associated with increased centromeric instability. Overall, these findings highlight the significance of Mycoplasma DnaK in the etiology of cancer and other genetic disorders providing a promising target for prevention, diagnostics, and therapeutics.

Mycoplasma DnaK increases DNA copy number variants in vivo.

The human microbiota affects critical cellular functions, although the responsible mechanism(s) is still poorly understood. In this regard, we previously showed that Mycoplasma fermentans DnaK, an HSP70 chaperone protein, hampers the activity of important cellular proteins responsible for DNA integrity. Here, we describe a novel DnaK knock-in mouse model generated in our laboratory to study the effect of M. fermentans DnaK expression in vivo. By using an array-based comparative genomic hybridization assay, we demonstrate that exposure to DnaK was associated with a higher number of DNA copy number variants (CNVs) indicative of unbalanced chromosomal alterations, together with reduced fertility and a high rate of fetal abnormalities. Consistent with their implication in genetic disorders, one of these CNVs caused a homozygous Grid2 deletion, resulting in an aberrant ataxic phenotype that recapitulates the extensive biallelic deletion in the Grid2 gene classified in humans as autosomal recessive spinocerebellar ataxia 18. Our data highlight a connection between components of the human urogenital tract microbiota, namely Mycoplasmas, and genetic abnormalities in the form of DNA CNVs, with obvious relevant medical, diagnostic, and therapeutic implications.

Engineering Escherichia coli for increased Und-P availability leads to material improvements in glycan expression technology.

BACKGROUND: Bacterial surface glycans are assembled by glycosyltransferases (GTs) that transfer sugar monomers to long-chained lipid carriers. Most bacteria employ the 55-carbon chain undecaprenyl phosphate (Und-P) to scaffold glycan assembly. The amount of Und-P available for glycan synthesis is thought to be limited by the rate of Und-P synthesis and by competition for Und-P between phosphoglycosyl transferases (PGTs) and GTs that prime glycan assembly (which we collectively refer to as PGT/GTs). While decreasing Und-P availability disrupts glycan synthesis and promotes cell death, less is known about the effects of increased Und-P availability. RESULTS: To determine if cells can maintain higher Und-P levels, we first reduced intracellular competition for Und-P by deleting all known non-essential PGT/GTs in the Gram-negative bacterium Escherichia coli (hereafter called ΔPGT/GT cells). We then increased the rate of Und-P synthesis in ΔPGT/GT cells by overexpressing the Und-P(P) synthase uppS from a plasmid (puppS). Und-P quantitation revealed that ΔPGT/GT/puppS cells can be induced to maintain 3-fold more Und-P than wild type cells. Next, we determined how increasing Und-P availability affects glycan expression. Interestingly, increasing Und-P availability increased endogenous and recombinant glycan expression. In particular, ΔPGT/GT/puppS cells could be induced to express 7-fold more capsule from Streptococcus pneumoniae serotype 4 than traditional E. coli cells used to express recombinant glycans. CONCLUSIONS: We demonstrate that the biotechnology standard bacterium E. coli can be engineered to maintain higher levels of Und-P. The results also strongly suggest that Und-P pathways can be engineered to increase the expression of potentially any Und-P-dependent polymer. Given that many bacterial glycans are central to the production of vaccines, diagnostics, and therapeutics, increasing Und-P availability should be a foremost consideration when designing bacterial glycan expression systems.

A genetic screen to identify factors affected by undecaprenyl phosphate recycling uncovers novel connections to morphogenesis in Escherichia coli.

Undecaprenyl phosphate (Und-P) is an essential lipid carrier that ferries cell wall intermediates across the cytoplasmic membrane in bacteria. Und-P is generated by dephosphorylating undecaprenyl pyrophosphate (Und-PP). In Escherichia coli, BacA, PgpB, YbjG, and LpxT dephosphorylate Und-PP and are conditionally essential. To identify vulnerabilities that arise when Und-P metabolism is defective, we developed a genetic screen for synthetic interactions which, in combination with ΔybjG ΔlpxT ΔbacA, are lethal or reduce fitness. The screen uncovered novel connections to cell division, DNA replication/repair, signal transduction, and glutathione metabolism. Further analysis revealed several new morphogenes; loss of one of these, qseC, caused cells to enlarge and lyse. QseC is the sensor kinase component of the QseBC two-component system. Loss of QseC causes overactivation of the QseB response regulator by PmrB cross-phosphorylation. Here, we show that deleting qseB completely reverses the shape defect of ΔqseC cells, as does overexpressing rprA (a small RNA). Surprisingly, deleting pmrB only partially suppressed qseC-related shape defects. Thus, QseB is activated by multiple factors in QseC's absence and prior functions ascribed to QseBC may originate from cell wall defects. Altogether, our findings provide a framework for identifying new determinants of cell integrity that could be targeted in future therapies.

Combined cART including Tenofovir Disoproxil, Emtricitabine, and Dolutegravir has potent therapeutic effects in HIV-1 infected humanized mice.

HIV-1 reservoirs persist in the presence of combined antiretroviral therapy (cART). However, cART has transformed HIV-1 infection into a chronic disease marked by control of HIV-1 viral load and mortality reduction. Major challenges remain, including viral resistance upon termination of cART and persistence and identification of tissue distribution of HIV-1 reservoirs. Thus, appropriate animal models that best mimic HIV-1 pathogenesis are important, and the current study complements our previously published validation of the CD34+ hematopoietic humanized mouse model for this purpose. Here we analyze viral suppression using the recently developed combination of antiretrovirals that include Tenofovir Disoproxil (TDF), Emtricitabine (FTC), and Dolutegravir (DTG), a choice based on recent clinical outcomes showing its improved antiretroviral potency, CD4+ T cell preservation, tolerability, and prevention of viral drug resistance compared to that of previous regimens. We used quantitative Airyscan-based super resolution confocal microscopy of selected mouse tissues. Our data allowed us to identify specific solid tissue reservoirs of human T cells expressing the HIV-1 core protein p24. In particular, lymph node, brain, spleen, and liver were visualized as reservoirs for residual infected cells. Marked reduction of viral replication was evident. Considering that detection and visualization of cryptic sites of HIV-1 infection in tissues are clearly crucial steps towards HIV-1 eradication, appropriate animal models with pseudo-human immune systems are needed. In fact, current studies with humans and non-human primates have limited sample availability at multiple stages of infection and cannot easily analyze the effects of differently administered combined antiretroviral treatments on multiple tissues. That is easier to manage when working with humanized mouse models, although we realize the limitations due to low human cell recovery and thus the number of cells available for thorough and comprehensive analyses. Nonetheless, our data further confirm that the CD34+ humanized mouse model is a potentially useful pre-clinical model to study and improve current anti-HIV-1 therapies.

Machine learning identifies ICU outcome predictors in a multicenter COVID-19 cohort.

BACKGROUND: Intensive Care Resources are heavily utilized during the COVID-19 pandemic. However, risk stratification and prediction of SARS-CoV-2 patient clinical outcomes upon ICU admission remain inadequate. This study aimed to develop a machine learning model, based on retrospective & prospective clinical data, to stratify patient risk and predict ICU survival and outcomes. METHODS: A Germany-wide electronic registry was established to pseudonymously collect admission, therapeutic and discharge information of SARS-CoV-2 ICU patients retrospectively and prospectively. Machine learning approaches were evaluated for the accuracy and interpretability of predictions. The Explainable Boosting Machine approach was selected as the most suitable method. Individual, non-linear shape functions for predictive parameters and parameter interactions are reported. RESULTS: 1039 patients were included in the Explainable Boosting Machine model, 596 patients retrospectively collected, and 443 patients prospectively collected. The model for prediction of general ICU outcome was shown to be more reliable to predict "survival". Age, inflammatory and thrombotic activity, and severity of ARDS at ICU admission were shown to be predictive of ICU survival. Patients' age, pulmonary dysfunction and transfer from an external institution were predictors for ECMO therapy. The interaction of patient age with D-dimer levels on admission and creatinine levels with SOFA score without GCS were predictors for renal replacement therapy. CONCLUSIONS: Using Explainable Boosting Machine analysis, we confirmed and weighed previously reported and identified novel predictors for outcome in critically ill COVID-19 patients. Using this strategy, predictive modeling of COVID-19 ICU patient outcomes can be performed overcoming the limitations of linear regression models. Trial registration "ClinicalTrials" (clinicaltrials.gov) under NCT04455451.

In vivo and in vitro cell-based model of lung adenocarcinoma from patients with pleural effusion.

Lung adenocarcinoma (LAC) is a common and aggressive form of lung cancer that is increasing in incidence among never smokers at a younger age. Current treatment of patients with LAC is insufficient and there is a need for identification of effective biomarkers and development of therapeutic targets. These demands require also improved models for in vivo and in vitro experimentation. In this study, we describe the establishment of two LAC cell lines, named LuCa-3 and LuCa-6. Both were derived from pleural effusion (PE) cells of LAC patients (L3 and L6) and readily propagated as tumor xenografts in immunodeficient mice. PE cells from the patient L6 exhibited also the capacity for in vitro growth and were cultured in two forms: (i) as a suspension growing cell population, labeled LuCa-6S, composed of non-clumping single cells; and (ii) as a monolayer-like culture, labeled LuCa-6A, exhibiting tight cell-to-cell and to culture surface adherence. Unique features of these two sublines and their cell clones are the capacity to convert from a non-clumping single-cell suspension into the adherent growth pattern and vice versa. Immunostaining of patients' tumor tissue xenografts and cultured subline cells displayed markers specific for the phenotype of human LAC. LuCa-6S and LuCa-6A cells did not reveal a noticeable disparity in quantitative growth characteristics. However, a number of differences were detected between these two cell populations manifested in detection or intensities of antigen expressions on the cell surface (CD133, SFTPC) and in the nucleus (TTF-1) including pluripotent (OCT-4, SOX-2, NANOG) genes in cancer stem-like cells (CSCs). Dissimilarities between these two sublines were also detected in N-glycan profiles and in the sensitivity to natural killer cells. Salient features of these subline cell populations are responsiveness to selective upregulation of the pluripotent genes in subsets of CSCs via conversion of their growth patterns and/or by using culture stem media with growth factors. The described in vivo/in vitro model enables broader experimental approaches in studies of lung adenocarcinoma.

Mycoplasma promotes malignant transformation in vivo, and its DnaK, a bacterial chaperone protein, has broad oncogenic properties.

We isolated a strain of human mycoplasma that promotes lymphomagenesis in SCID mice, pointing to a p53-dependent mechanism similar to lymphomagenesis in uninfected p53-/- SCID mice. Additionally, mycoplasma infection in vitro reduces p53 activity. Immunoprecipitation of p53 in mycoplasma-infected cells identified several mycoplasma proteins, including DnaK, a member of the Hsp70 chaperon family. We focused on DnaK because of its ability to interact with proteins. We demonstrate that mycoplasma DnaK interacts with and reduces the activities of human proteins involved in critical cellular pathways, including DNA-PK and PARP1, which are required for efficient DNA repair, and binds to USP10 (a key p53 regulator), impairing p53-dependent anticancer functions. This also reduced the efficacy of anticancer drugs that depend on p53 to exert their effect. mycoplasma was detected early in the infected mice, but only low copy numbers of mycoplasma DnaK DNA sequences were found in some primary and secondary tumors, pointing toward a hit-and-run/hide mechanism of transformation. Uninfected bystander cells took up exogenous DnaK, suggesting a possible paracrine function in promoting malignant transformation, over and above cells infected with the mycoplasma. Phylogenetic amino acid analysis shows that other bacteria associated with human cancers have similar DnaKs, consistent with a common mechanism of cellular transformation mediated through disruption of DNA-repair mechanisms, as well as p53 dysregulation, that also results in cancer-drug resistance. This suggests that the oncogenic properties of certain bacteria are DnaK-mediated.

The Endocannabinoid System: A Potential Target for the Treatment of Various Diseases.

The Endocannabinoid System (ECS) is primarily responsible for maintaining homeostasis, a balance in internal environment (temperature, mood, and immune system) and energy input and output in living, biological systems. In addition to regulating physiological processes, the ECS directly influences anxiety, feeding behaviour/appetite, emotional behaviour, depression, nervous functions, neurogenesis, neuroprotection, reward, cognition, learning, memory, pain sensation, fertility, pregnancy, and pre-and post-natal development. The ECS is also involved in several pathophysiological diseases such as cancer, cardiovascular diseases, and neurodegenerative diseases. In recent years, genetic and pharmacological manipulation of the ECS has gained significant interest in medicine, research, and drug discovery and development. The distribution of the components of the ECS system throughout the body, and the physiological/pathophysiological role of the ECS-signalling pathways in many diseases, all offer promising opportunities for the development of novel cannabinergic, cannabimimetic, and cannabinoid-based therapeutic drugs that genetically or pharmacologically modulate the ECS via inhibition of metabolic pathways and/or agonism or antagonism of the receptors of the ECS. This modulation results in the differential expression/activity of the components of the ECS that may be beneficial in the treatment of a number of diseases. This manuscript in-depth review will investigate the potential of the ECS in the treatment of various diseases, and to put forth the suggestion that many of these secondary metabolites of Cannabis sativa L. (hereafter referred to as "C. sativa L." or "medical cannabis"), may also have potential as lead compounds in the development of cannabinoid-based pharmaceuticals for a variety of diseases.

Antiviral Activity of Jamaican Medicinal Plants and Isolated Bioactive Compounds.

Plants have had historical significance in medicine since the beginning of civilization. The oldest medical pharmacopeias of the African, Arabian, and Asian countries solely utilize plants and herbs to treat pain, oral diseases, skin diseases, microbial infections, multiple types of cancers, reproductive disorders among a myriad of other ailments. The World Health Organization (WHO) estimates that over 65% of the world population solely utilize botanical preparations as medicine. Due to the abundance of plants, plant-derived medicines are more readily accessible, affordable, convenient, and have safer side-effect profiles than synthetic drugs. Plant-based decoctions have been a significant part of Jamaican traditional folklore medicine. Jamaica is of particular interest because it has approximately 52% of the established medicinal plants that exist on earth. This makes the island particularly welcoming for rigorous scientific research on the medicinal value of plants and the development of phytomedicine thereof. Viral infections caused by the human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2), hepatitis virus B and C, influenza A virus, and the severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) present a significant global burden. This is a review of some important Jamaican medicinal plants, with particular reference to their antiviral activity.

Simultaneously inhibiting undecaprenyl phosphate production and peptidoglycan synthases promotes rapid lysis in Escherichia coli.

Peptidoglycan (PG) is a highly cross-linked polysaccharide that encases bacteria, resists the effects of turgor and confers cell shape. PG precursors are translocated across the cytoplasmic membrane by the lipid carrier undecaprenyl phosphate (Und-P) where they are incorporated into the PG superstructure. Previously, we found that one of our Escherichia coli laboratory strains (CS109) harbors a missense mutation in uppS, which encodes an enzymatically defective Und-P(P) synthase. Here, we show that CS109 cells lacking the bifunctional aPBP PBP1B (penicillin binding protein 1B) lyse during exponential growth at elevated temperature. PBP1B lysis was reversed by: (i) reintroducing wild-type uppS, (ii) increasing the availability of PG precursors or (iii) overproducing PBP1A, a related bifunctional PG synthase. In addition, inhibiting the catalytic activity of PBP2 or PBP3, two monofunctional bPBPs, caused CS109 cells to lyse. Limiting the precursors required for Und-P synthesis in MG1655, which harbors a wild-type allele of uppS, also promoted lysis in mutants lacking PBP1B or bPBP activity. Thus, simultaneous inhibition of Und-P production and PG synthases provokes a synergistic response that leads to cell lysis. These findings suggest a biological connection that could be exploited in combination therapies.

N-Glycan profiling of lung adenocarcinoma in patients at different stages of disease.

Lung adenocarcinoma (LAC) is the most common form of lung cancer that increases in non-smokers at younger age. Altered protein glycosylation is one of the hallmarks of malignancy, its role in cancer progression is still poorly understood. In this study, we report mass spectrometric (MS) analysis of N-glycans released from fresh or defrosted tissue specimens from 24 patients with LAC. Comparison of cancerous versus adjacent healthy tissues revealed substantial differences in N-glycan profiles associated with disease. The significant increase in paucimannose and high-mannose glycans with 6-9 mannose residues and decline in the sialylated complex biantenary core fucosylated glycan with composition NeuAcGal2GlcNAc2Man3GlcNAc2Fuc were general features of tumors. In addition, 42 new N-glycan compositions were detected in cancerous tissues. The prominent changes in advanced disease stages were mostly observed in core fucosylated N-glycans with additional fucose (Fuc) residue/s and enhanced branching with non-galactosylated N-acetyl-glucosamine (GlcNAc) units. Both of these monosaccharide types were linked preferably on the 6-antenna. Importantly, as compared with noncancerous tissues, a number of these significant changes were clearly detectable early on in stage I. Application of N-glycan data obtained from tissues was next assessed and validated for evaluation of small sized biopsies obtained via bronchoscopy. In summary, observed alterations and data of newly detected N-glycans expand knowledge about the glycosylation in LAC and may contribute to research in more tailored therapies. Moreover, the results demonstrate effectiveness of the presented approach for utility in rapid discrimination of cancerous from healthy lung tissues.

Intersecting roles of ER stress, mitochondrial dysfunction, autophagy, and calcium homeostasis in HIV-associated neurocognitive disorder.

HIV-associated neurocognitive disorder (HAND) is a collective term describing the spectrum of neurocognitive deficits that arise from HIV infection. Although the introduction to highly active antiretroviral therapy (HAART) has prolonged the lifespan of HIV patients, neurocognitive impairments remain prevalent, as patients are left perpetually with HIV. Currently, physicians face a challenge in treating HAND patients, so a greater understanding of the mechanisms underlying HAND pathology has been a growing focus in HIV research. Recent research has revealed the role disrupted calcium homeostasis in HIV-mediated neurotoxicity. Calcium plays a well-established role in the crosstalk between the mitochondrion and ER as well as in regulating autophagy, and ER stress, mitochondrial dysfunction, and impaired autophagic activity are considered hallmarks in several neurodegenerative and neurocognitive disorders. Therefore, it is paramount that the intricate inter-organelle signaling in relation to calcium homeostasis during HIV infection and the development of HAND is elucidated. This review consolidates current knowledge regarding the neuropathology of neurocognitive disorders and HIV infection with a focus on the underlying role of calcium during ER stress, mitochondrial dysfunction, and autophagy associated with the progression of HAND. The details of this intricate crosstalk during HAND remain relatively unknown; further research in this field can potentially aid in the development of improved therapy for patients suffering from HAND.

Role of the inflammasomes in HIV-associated neuroinflammation and neurocognitive disorders.

HIV associated neurocognitive disorders (HAND) is a unique form of neurological impairment that stems from HIV. This disease and its characteristics can be accredited to incorporation of DNA and mRNA of HIV-1 into the CNS. A proper understanding of the intricacies of HAND and the underlying mechanisms associated with corresponding immune reactions are vital for the potential development of a reliable treatment for HAND. A common phenomenon observed in CNS cells, specifically microglia, that are infected with HAND is inflammation, which is a consequence of the activation of innate immune response due to a variety of stimuli, in this case, being the HIV infection. The CNS based inflammation is mediated by the production of cytokines, chemokines, reactive oxygen species, and secondary messengers, which occurs at CNS glia, endothelial cells and peripherally derived immune cells. Inflammasomes play a significant role with regard to neuroinflammation due to their ability to dictate the activation of various inflammatory responses. Certain stimuli can result in the activation of caspase-1; hence, leading to the processing of interleukin-1ß and interleukin-18 pro-inflammatory cytokines. The processed IL-1ß and IL-18 activate signaling pathways that begin the process of neuroinflammation. Due to the fact that the NLRP3 inflammasome is the most abundant in the CNS, it is the most extensively investigated inflammasome with regard to the nervous system. Due to the importance of neuroinflammation in the evolution of HAND and proliferation of neuroinflammation due to HAND, it can be concluded that there exists a relationship between HAND and inflammasomes. The aim of our review is to consolidate current knowledge of important mechanisms in HAND, specifically related to its relationship with neuroinflammation and inflammasomes to shed light on a possible improved treatment for HAND.

HIVAN associated tubular pathology with reference to ER stress, mitochondrial changes, and autophagy.

Human immunodeficiency virus associated nephropathy (HIVAN) is a unique form of a renal parenchymal disorder. This disease and its characteristics can be accredited to incorporation of DNA and mRNA of human immunodeficiency virus type 1 into the renal parenchymal cells. A proper understanding of the intricacies of HIVAN and the underlying mechanisms associated with renal function and disorders is vital for the potential development of a reliable treatment for HIVAN. Specifically, the renal tubule segment of the kidney is characterized by its transport capabilities and its ability to reabsorb water and salts into the blood. However, the segment is also known for certain disorders, such as renal tubular epithelial cell infection and microcyst formation, which are also closely linked to HIVAN. Furthermore, certain organelles, like the endoplasmic reticulum (ER), mitochondria, and lysosome, are vital for certain underlying mechanisms in kidney cells. A paradigm of the importance of said organelles can be seen in documented cases of HIVAN where the renal disorder results increased ER stress due to HIV viral propagation. This balance can be restored through the synthesis of secretory proteins, but, in return, the secretion requires more energy; therefore, there is a noticeable increase in mitochondrial stress. The increased ER changes and mitochondrial stress will greatly upregulate the process of autophagy, which involves the cell's lysosomes. In conjunction, we found that ER stress and mitochondrial changes are associated in the Tg26 animal model of HIVAN. The aim of our review is to consolidate current knowledge of important mechanisms in HIVAN, specifically related to the renal tubules' association with ER stress, mitochondrial changes and autophagy. Although the specific regulatory mechanism detailing the cross-talk between the various organelles is unknown in HIVAN, the continued research in this field may potentially shed light on a possible improved treatment for HIVAN.

Expression of HIV-1 matrix protein p17 and association with B-cell lymphoma in HIV-1 transgenic mice.

HIV-1 infection is associated with increased risk for B-cell lymphomas. How HIV infection promotes the development of lymphoma is unclear, but it may involve chronic B-cell activation, inflammation, and/or impaired immunity, possibly leading to a loss of control of oncogenic viruses and reduced tumor immunosurveillance. We hypothesized that HIV structural proteins may contribute to lymphomagenesis directly, because they can persist long term in lymph nodes in the absence of viral replication. The HIV-1 transgenic mouse Tg26 carries a noninfectious HIV-1 provirus lacking part of the gag-pol region, thus constituting a model for studying the effects of viral products in pathogenesis. Approximately 15% of Tg26 mice spontaneously develop leukemia/lymphoma. We investigated which viral proteins are associated with the development of leukemia/lymphoma in the Tg26 mouse model, and performed microarray analysis on RNA from spleen and lymph nodes to identify potential mechanisms of lymphomagenesis. Of the viral proteins examined, only expression of HIV-1 matrix protein p17 was associated with leukemia/lymphoma development and was highly expressed in bone marrow before disease. The tumor cells resembled pro-B cells, and were CD19+IgM-IgD-CD93+CD43+CD21-CD23-VpreB+CXCR4+ Consistent with the pro-B-cell stage of B-cell development, microarray analysis revealed enrichment of transcripts, including Rag1, Rag2, CD93, Vpreb1, Vpreb3, and Igll1 We confirmed RAG1 expression in Tg26 tumors, and hypothesized that HIV-1 matrix protein p17 may directly induce RAG1 in B cells. Stimulation of human activated B cells with p17 enhanced RAG1 expression in three of seven donors, suggesting that intracellular signaling by p17 may lead to genomic instability and transformation.

Comparative analysis of FKBP family protein: evaluation, structure, and function in mammals and Drosophila melanogaster.

BACKGROUND: FK506-binding proteins (FKBPs) have become the subject of considerable interest in several fields, leading to the identification of several cellular and molecular pathways in which FKBPs impact prenatal development and pathogenesis of many human diseases. MAIN BODY: This analysis revealed differences between how mammalian and Drosophila FKBPs mechanisms function in relation to the immunosuppressant drugs, FK506 and rapamycin. Differences that could be used to design insect-specific pesticides. (1) Molecular phylogenetic analysis of FKBP family proteins revealed that the eight known Drosophila FKBPs share homology with the human FKBP12. This indicates a close evolutionary relationship, and possible origination from a common ancestor. (2) The known FKBPs contain FK domains, that is, a prolyl cis/trans isomerase (PPIase) domain that mediates immune suppression through inhibition of calcineurin. The dFKBP59, CG4735/Shutdown, CG1847, and CG5482 have a Tetratricopeptide receptor domain at the C-terminus, which regulates transcription and protein transportation. (3) FKBP51 and FKBP52 (dFKBP59), along with Cyclophilin 40 and protein phosphatase 5, function as Hsp90 immunophilin co-chaperones within steroid receptor-Hsp90 heterocomplexes. These immunophilins are potential drug targets in pathways associated with normal physiology and may be used to treat a variety of steroid-based diseases by targeting exocytic/endocytic cycling and vesicular trafficking. (4) By associating with presinilin, a critical component of the Notch signaling pathway, FKBP14 is a downstream effector of Notch activation at the membrane. Meanwhile, Shutdown associates with transposons in the PIWI-interacting RNA pathway, playing a crucial role in both germ cells and ovarian somas. Mutations in or silencing of dFKBPs lead to early embryonic lethality in Drosophila. Therefore, further understanding the mechanisms of FK506 and rapamycin binding to immunophilin FKBPs in endocrine, cardiovascular, and neurological function in both mammals and Drosophila would provide prospects in generating unique, insect specific therapeutics targeting the above cellular signaling pathways. CONCLUSION: This review will evaluate the functional roles of FKBP family proteins, and systematically summarize the similarities and differences between FKBP proteins in Drosophila and Mammals. Specific therapeutics targeting cellular signaling pathways will also be discussed.

Cigarette smoke and nicotine effects on brain proinflammatory responses and behavioral and motor function in HIV-1 transgenic rats.

Cognitive impairment in HIV-1 infection is associated with the induction of chronic proinflammatory responses in the brains of infected individuals. The risk of HIV-related cognitive impairment is increased by cigarette smoking, which induces brain inflammation in rodent models. To better understand the role of smoking and the associated immune response on behavioral and motor function in HIV infection, wild-type F344 and HIV-1 transgenic (HIV1Tg) rats were exposed to either smoke from nicotine-containing (regular) cigarettes, smoke from nicotine-free cigarettes, or to nicotine alone. The animals were then tested using the rotarod test (RRT), the novel object recognition test (NORT), and the open field test (OFT). Subsequently, brain frontal cortex from the rats was analyzed for levels of TNF-α, IL-1, and IL-6. On the RRT, impairment was noted for F344 rats exposed to either nicotine-free cigarette smoke or nicotine alone and for F344 and HIV1Tg rats exposed to regular cigarette smoke. Effects from the exposures on the OFT were seen only for HIV1Tg rats, for which function was worse following exposure to regular cigarette smoke as compared to exposure to nicotine alone. Expression levels for all three cytokines were overall higher for HIV1Tg than for F344 rats. For HIV1Tg rats, TNF-α, IL-1, and IL-6 gene expression levels for all exposure groups were higher than for control rats. All F344 rat exposure groups also showed significantly increased TNF-α expression levels. However, for F344 rats, IL-1 expression levels were higher only for rats exposed to nicotine-free and nicotine-containing CS, and no increase in IL-6 gene expression was noted with any of the exposures as compared to controls. These studies, therefore, demonstrate that F344 and HIV1Tg rats show differential behavioral and immune effects from these exposures. These effects may potentially reflect differences in the responsiveness of the various brain regions in the two animal species as well as the result of direct toxicity mediated by the proinflammatory cytokines that are produced by HIV proteins and by other factors that are present in regular cigarette smoke.

Glomerular mitochondrial changes in HIV associated renal injury.

HIV-associated nephropathy (HIVAN) is an AIDs-related disease of the kidney. HIVAN is characterized by severe proteinuria, podocyte hyperplasia, collapse, glomerular, and tubulointerstitial damage. HIV-1 transgenic (Tg26) mouse is the most popular model to study the HIV manifestations that develop similar renal presentations as HIVAN. Viral proteins, including Tat, Nef, and Vpr play a significant role in renal cell damage. It has been shown that mitochondrial changes are involved in several kidney diseases, and therefore, mitochondrial dysfunction may be implicated in the pathology of HIVAN. In the present study, we investigated the changes of mitochondrial homeostasis, biogenesis, dynamics, mitophagy, and examined the role of reactive oxygen species (ROS) generation and apoptosis in the Tg26 mouse model. The Tg26 mice showed significant impairment of kidney function, which was accompanied by increased blood urea nitrogen (BUN), creatinine and protein urea level. In addition, histological, western blot and PCR analysis of the Tg26 mice kidneys showed a downregulation of NAMPT, SIRT1, and SIRT3 expressions levels. Furthermore, the kidney of the Tg26 mice showed a downregulation of PGC1α, MFN2, and PARKIN, which are coupled with decrease of mitochondrial biogenesis, imbalance of mitochondrial dynamics, and downregulation of mitophagy, respectively. Furthermore, our results indicate that mitochondrial dysfunction were associated with ER stress, ROS generation and apoptosis. These results strongly suggest that the impaired mitochondrial morphology, homeostasis, and function associated with HIVAN. These findings indicated that a new insight on pathological mechanism associated with mitochondrial changes in HIVAN and a potential therapeutic target.

Renal aquaporin-4 associated pathology in TG-26 mice.

Human immunodeficiency virus-associated nephropathy (HIVAN) is a leading cause of end-stage renal disease in HIV patients, which is characterized by glomerulosclerosis and renal tubular dysfunction. Aquaporin-4 (AQP-4) is a membrane bound water channel protein that plays a distinct role in water reabsorption from renal tubular fluid. It has been proven that failure of AQP-4 insertion into the renal tubular membrane leads to renal dysfunction. However, the role of AQP-4 in HIVAN is unclear. We hypothesize that impaired water reabsorption leads to renal injury in HIVAN, where AQP-4 plays a crucial role. Renal function is assessed by urinary protein and serum blood urea nitrogen (BUN). Kidneys from HIV Transgenic (TG26) mice (HIVAN animal model) were compared to wild type mice by immunostaining, immunoblotting and quantitative RT-PCR. TG26 mice had increased proteinuria and BUN. We found decreased AQP-4 levels in the renal medulla, increased endothelin-1, endothelin receptor A and reduced Sirtuin1 (SIRT-1) levels in TG26 mice. Also, oxidative and endoplasmic reticulum stress was enhanced in kidneys of TG26 mice. We provide the first evidence that AQP-4 is inhibited due to induction of HIV associated stress in the kidneys of TG26 mice which limits water reabsorption in the kidney which may be one of the cause associated with HIVAN, impairing kidney physiology. AQP-4 dysregulation in TG26 mice suggests that similar changes may occur in HIVAN patients. This work may identify new therapeutic targets to be evaluated in HIVAN.