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.

Functional neuropathology of neonatal hypoxia-ischemia by single-mouse longitudinal electroencephalography.

OBJECTIVE: Neonatal cerebral hypoxia-ischemia (HI) results in symptomatic seizures and long-term neurodevelopmental disability. The Rice-Vannucci model of rodent neonatal HI has been used extensively to examine and translate the functional consequences of acute and chronic HI-induced encephalopathy. Yet, longitudinal electrophysiological characterization of this brain injury model has been limited by the size of the neonatal mouse's head and postnatal maternal dependency. We overcome this challenge by employing a novel method of longitudinal single-mouse electroencephalography (EEG) using chronically implanted subcranial electrodes in the term-equivalent mouse pup. We characterize the neurophysiological disturbances occurring during awake and sleep states in the acute and chronic phases following newborn brain injury. METHODS: C57BL/6 mice underwent long-term bilateral subcranial EEG and electromyographic electrode placement at postnatal day 9 followed by unilateral carotid cauterization and exposure to 40 minutes of hypoxia the following day. EEG recordings were obtained prior, during, and intermittently after the HI procedure from postnatal day 10 to weaning age. Quantitative EEG and fast Fourier transform analysis were used to evaluate seizures, cortical cerebral dysfunction, and disturbances in vigilance states. RESULTS: We observed neonatal HI-provoked electrographic focal and bilateral seizures during or immediately following global hypoxia and most commonly contralateral to the ischemic injury. Spontaneous chronic seizures were not seen. Injured mice developed long-term asymmetric EEG background attenuation in all frequencies and most prominently during non-rapid eye movement (NREM) sleep. HI mice also showed transient impairments in vigilance state duration and transitions during the first 2 days following injury. SIGNIFICANCE: The functional burden of mouse neonatal HI recorded by EEG resembles closely that of the injured human newborn. The use of single-mouse longitudinal EEG in this immature model can advance our understanding of the developmental and pathophysiological mechanisms of neonatal cerebral injury and help translate novel therapeutic strategies against this devastating condition.

The Chromatin Remodeler HELLS: A New Regulator in DNA Repair, Genome Maintenance, and Cancer.

Robust, tightly regulated DNA repair is critical to maintaining genome stability and preventing cancer. Eukaryotic DNA is packaged into chromatin, which has a profound, yet incompletely understood, regulatory influence on DNA repair and genome stability. The chromatin remodeler HELLS (helicase, lymphoid specific) has emerged as an important epigenetic regulator of DNA repair, genome stability, and multiple cancer-associated pathways. HELLS belongs to a subfamily of the conserved SNF2 ATP-dependent chromatin-remodeling complexes, which use energy from ATP hydrolysis to alter nucleosome structure and packaging of chromatin during the processes of DNA replication, transcription, and repair. The mouse homologue, LSH (lymphoid-specific helicase), plays an important role in the maintenance of heterochromatin and genome-wide DNA methylation, and is crucial in embryonic development, gametogenesis, and maturation of the immune system. Human HELLS is abundantly expressed in highly proliferating cells of the lymphoid tissue, skin, germ cells, and embryonic stem cells. Mutations in HELLS cause the human immunodeficiency syndrome ICF (Immunodeficiency, Centromeric instability, Facial anomalies). HELLS has been implicated in many types of cancer, including retinoblastoma, colorectal cancer, hepatocellular carcinoma, and glioblastoma. Here, we review and summarize accumulating evidence highlighting important roles for HELLS in DNA repair, genome maintenance, and key pathways relevant to cancer development, progression, and treatment.

The role of the histone H3 variant CENPA in prostate cancer.

Overexpression of centromeric proteins has been identified in a number of human malignancies, but the functional and mechanistic contributions of these proteins to disease progression have not been characterized. The centromeric histone H3 variant centromere protein A (CENPA) is an epigenetic mark that determines centromere identity. Here, using an array of approaches, including RNA-sequencing and ChIP-sequencing analyses, immunohistochemistry-based tissue microarrays, and various cell biology assays, we demonstrate that CENPA is highly overexpressed in prostate cancer in both tissue and cell lines and that the level of CENPA expression correlates with the disease stage in a large cohort of patients. Gain-of-function and loss-of-function experiments confirmed that CENPA promotes prostate cancer cell line growth. The results from the integrated sequencing experiments suggested a previously unidentified function of CENPA as a transcriptional regulator that modulates expression of critical proliferation, cell-cycle, and centromere/kinetochore genes. Taken together, our findings show that CENPA overexpression is crucial to prostate cancer growth.

Rapid molecular assays to study human centromere genomics.

The centromere is the structural unit responsible for the faithful segregation of chromosomes. Although regulation of centromeric function by epigenetic factors has been well-studied, the contributions of the underlying DNA sequences have been much less well defined, and existing methodologies for studying centromere genomics in biology are laborious. We have identified specific markers in the centromere of 23 of the 24 human chromosomes that allow for rapid PCR assays capable of capturing the genomic landscape of human centromeres at a given time. Use of this genetic strategy can also delineate which specific centromere arrays in each chromosome drive the recruitment of epigenetic modulators. We further show that, surprisingly, loss and rearrangement of DNA in centromere 21 is associated with trisomy 21. This new approach can thus be used to rapidly take a snapshot of the genetics and epigenetics of each specific human centromere in nondisjunction disorders and other biological settings.

Early developmental electroencephalography abnormalities, neonatal seizures, and induced spasms in a mouse model of tuberous sclerosis complex.

OBJECTIVE: Tuberous sclerosis complex (TSC) is one of the most common genetic causes of epilepsy. Seizures in TSC typically first present in infancy or early childhood, including focal seizures and infantile spasms. Infantile spasms in TSC are particularly characteristic in its strong responsiveness to vigabatrin. Although a number of mouse models of epilepsy in TSC have been described, there are very limited electroencephalographic (EEG) or seizure data during the preweanling neonatal and infantile-equivalent mouse periods. Tsc1GFAP CKO mice are a well-characterized mouse model of epilepsy in TSC, but whether these mice have seizures during early development has not been documented. The objective of this study was to determine whether preweanling Tsc1GFAP CKO mice have developmental EEG abnormalities or seizures, including spasms. METHODS: Longitudinal video-EEG and electromyographic recordings were performed serially on Tsc1GFAP CKO and control mice from postnatal days 9-21 and analyzed for EEG background abnormalities, sleep-wake vigilance states, and spontaneous seizures. Spasms were also induced with varying doses of N-methyl-D-aspartate (NMDA). RESULTS: The interictal EEG of Tsc1GFAP CKO mice had excessive discontinuity and slowing, suggesting a delayed developmental progression compared with control mice. Tsc1GFAP CKO mice also had increased vigilance state transitions and fragmentation. Tsc1GFAP CKO mice had spontaneous focal seizures in the early neonatal period and a reduced threshold for NMDA-induced spasms, but no spontaneous spasms were observed. SIGNIFICANCE: Neonatal Tsc1GFAP CKO mice recapitulate early developmental aspects of EEG abnormalities, focal seizures, and an increased propensity for spasms. This mouse model may be useful for early mechanistic and therapeutic studies of epileptogenesis in TSC.

Susceptibility of Human Endogenous Retrovirus Type K to Reverse Transcriptase Inhibitors.

Human endogenous retroviruses (HERVs) make up 8% of the human genome. The HERV type K (HERV-K) HML-2 (HK2) family contains proviruses that are the most recent entrants into the human germ line and are transcriptionally active. In HIV-1 infection and cancer, HK2 genes produce retroviral particles that appear to be infectious, yet the replication capacity of these viruses and potential pathogenicity has been difficult to ascertain. In this report, we screened the efficacy of commercially available reverse transcriptase inhibitors (RTIs) at inhibiting the enzymatic activity of HK2 RT and HK2 genomic replication. Interestingly, only one provirus, K103, was found to encode a functional RT among those examined. Several nucleoside analogue RTIs (NRTIs) blocked K103 RT activity and consistently inhibited the replication of HK2 genomes. The NRTIs zidovudine (AZT), stavudine (d4T), didanosine (ddI), and lamivudine (3TC), and the nucleotide RTI inhibitor tenofovir (TDF), show efficacy in blocking K103 RT. HIV-1-specific nonnucleoside RTIs (NNRTIs), protease inhibitors (PIs), and integrase inhibitors (IIs) did not affect HK2, except for the NNRTI etravirine (ETV). The inhibition of HK2 infectivity by NRTIs appears to take place at either the reverse transcription step of the viral genome prior to HK2 viral particle formation and/or in the infected cells. Inhibition of HK2 by these drugs will be useful in suppressing HK2 infectivity if these viruses prove to be pathogenic in cancer, neurological disorders, or other diseases associated with HK2. The present studies also elucidate a key aspect of the life cycle of HK2, specifically addressing how they do, and/or did, replicate.IMPORTANCE Endogenous retroviruses are relics of ancestral virus infections in the human genome. The most recent of these infections was caused by HK2. While HK2 often remains silent in the genome, this group of viruses is activated in HIV-1-infected and cancer cells. Recent evidence suggests that these viruses are infectious, and the potential exists for HK2 to contribute to disease. We show that HK2, and specifically the enzyme that mediates virus replication, can be inhibited by a panel of drugs that are commercially available. We show that several drugs block HK2 with different efficacies. The inhibition of HK2 replication by antiretroviral drugs appears to occur in the virus itself as well as after infection of cells. Therefore, these drugs might prove to be an effective treatment by suppressing HK2 infectivity in diseases where these viruses have been implicated, such as cancer and neurological syndromes.

HIV infection reveals widespread expansion of novel centromeric human endogenous retroviruses.

Human endogenous retroviruses (HERVs) make up 8% of the human genome. The HERV-K (HML-2) family is the most recent group of these viruses to have inserted into the genome, and we have detected the activation of HERV-K (HML-2) proviruses in the blood of patients with HIV-1 infection. We report that HIV-1 infection activates expression of a novel HERV-K (HML-2) provirus, termed K111, present in multiple copies in the centromeres of chromosomes throughout the human genome yet not annotated in the most recent human genome assembly. Infection with HIV-1 or stimulation with the HIV-1 Tat protein leads to the activation of K111 proviruses. K111 is present as a single copy in the genome of the chimpanzee, yet K111 is not found in the genomes of other primates. Remarkably, K111 proviruses appear in the genomes of the extinct Neanderthal and Denisovan, while modern humans have at least 100 K111 proviruses spread across the centromeres of 15 chromosomes. Our studies suggest that the progenitor K111 integrated before the Homo-Pan divergence and expanded in copy number during the evolution of hominins, perhaps by recombination. The expansion of K111 provides sequence evidence suggesting that recombination between the centromeres of various chromosomes took place during the evolution of humans. K111 proviruses show significant sequence variations in each individual centromere, which may serve as markers in future efforts to annotate human centromere sequences. Further, this work is an example of the potential to discover previously unknown genomic sequences through the analysis of nucleic acids found in the blood of patients.

Human Endogenous Retrovirus Type K (HERV-K) Particles Package and Transmit HERV-K-Related Sequences.

UNLABELLED: Human endogenous retroviruses (HERV) make up 8% of the human genome. While the youngest of these retroviruses, HERV-K(HML-2), termed HK2, is able to code for all viral proteins and produce virus-like particles, it is not known if these virus particles package and transmit HK2-related sequences. Here, we analyzed the capacity of HK2 for packaging and transmitting HK2 sequences. We created an HK2 probe, termed Bogota, which can be packaged into HK2 viruses, and transfected it into cells that make HK2 particles. Supernatants of the transfected cells, which contained HK2 viral particles, then were added to target cells, and the transmissibility of the HK2 Bogota reporter was tracked by G418 resistance. Our studies revealed that contemporary HK2 virions produced by some teratocarcinoma and breast cancer cell lines, as well as by peripheral blood lymphocytes from lymphoma patients, can package HK2 Bogota probes, and these viruses transmitted these probes to other cells. After transmission, HK2 Bogota transcripts undergo reverse transcription, a step impaired by antiretroviral agents or by introduction of mutations into the probe sequences required for reverse transcription. HK2 viruses were more efficiently transmitted in the presence of HK2 Rec or HIV-1 Tat and Vif. Transmitted Bogota probes formed episomes but did not integrate into the cellular genome. Resistance to integration might explain the relatively low number of HK2 insertions that were acquired during the last 25 million years of evolution. Whether transient transmission of modern HK2 sequences, which encode two putative oncoproteins, can lead to disease remains to be studied. IMPORTANCE: Retroviruses invaded the genome of human ancestors over the course of millions of years, yet these viruses generally have been inactivated during evolution, with only remnants of these infectious sequences remaining in the human genome. One of these viruses, termed HK2, still is capable of producing virus particles, although these particles have been regarded as being noninfectious. Using a genetic probe derived from HK2, we have discovered that HK2 viruses produced in modern humans can package HK2 sequences and transmit them to various other cells. Furthermore, the genetic sequences packaged in HK2 undergo reverse transcription. The transmitted probe circularized in the cell and failed to integrate into the cellular genome. These findings suggest that modern HK2 viruses can package viral RNA and transmit it to other cells. Contrary to previous views, we provide evidence of an extracellular viral phase of modern HK2 viruses. We have no evidence of sustained, spreading infection.

Genomic flexibility of human endogenous retrovirus type K.

UNLABELLED: Human endogenous retrovirus type K (HERV-K) proviruses are scattered throughout the human genome, but as no infectious HERV-K virus has been detected to date, the mechanism by which these viruses replicated and populated the genome remains unresolved. Here, we provide evidence that, in addition to the RNA genomes that canonical retroviruses package, modern HERV-K viruses can contain reverse-transcribed DNA (RT-DNA) genomes. Indeed, reverse transcription of genomic HERV-K RNA into the DNA form is able to occur in three distinct times and locations: (i) in the virus-producing cell prior to viral release, yielding a DNA-containing extracellular virus particle similar to the spumaviruses; (ii) within the extracellular virus particle itself, transitioning from an RNA-containing particle to a DNA-containing particle; and (iii) after entry of the RNA-containing virus into the target cell, similar to canonical retroviruses, such as murine leukemia virus and HIV. Moreover, using a resuscitated HERV-K virus construct, we show that both viruses with RNA genomes and viruses with DNA genomes are capable of infecting target cells. This high level of genomic flexibility historically could have permitted these viruses to replicate in various host cell environments, potentially assisting in their many integration events and resulting in their high prevalence in the human genome. Moreover, the ability of modern HERV-K viruses to proceed through reverse transcription and package RT-DNA genomes suggests a higher level of replication competency than was previously understood, and it may be relevant in HERV-K-associated human diseases. IMPORTANCE: Retroviral elements comprise at least 8% of the human genome. Of all the endogenous retroviruses, HERV-K viruses are the most intact and biologically active. While a modern infectious HERV-K has yet to be found, HERV-K activation has been associated with cancers, autoimmune diseases, and HIV-1 infection. Thus, determining how this virus family became such a prevalent member of our genome and what it is capable of in its current form are of the utmost importance. Here, we provide evidence that HERV-K viruses currently found in the human genome are able to proceed through reverse transcription and historically utilized a life cycle with a surprising degree of genomic flexibility in which both RNA- and DNA-containing viruses were capable of mediating infection.

Regulation of the human endogenous retrovirus K (HML-2) transcriptome by the HIV-1 Tat protein.

UNLABELLED: Approximately 8% of the human genome is made up of endogenous retroviral sequences. As the HIV-1 Tat protein activates the overall expression of the human endogenous retrovirus type K (HERV-K) (HML-2), we used next-generation sequencing to determine which of the 91 currently annotated HERV-K (HML-2) proviruses are regulated by Tat. Transcriptome sequencing of total RNA isolated from Tat- and vehicle-treated peripheral blood lymphocytes from a healthy donor showed that Tat significantly activates expression of 26 unique HERV-K (HML-2) proviruses, silences 12, and does not significantly alter the expression of the remaining proviruses. Quantitative reverse transcription-PCR validation of the sequencing data was performed on Tat-treated PBLs of seven donors using provirus-specific primers and corroborated the results with a substantial degree of quantitative similarity. IMPORTANCE: The expression of HERV-K (HML-2) is tightly regulated but becomes markedly increased following infection with HIV-1, in part due to the HIV-1 Tat protein. The findings reported here demonstrate the complexity of the genome-wide regulation of HERV-K (HML-2) expression by Tat. This work also demonstrates that although HERV-K (HML-2) proviruses in the human genome are highly similar in terms of DNA sequence, modulation of the expression of specific proviruses in a given biological situation can be ascertained using next-generation sequencing and bioinformatics analysis.

Human chorionic gonadotropin decreases cerebral cystic encephalomalacia and parvalbumin interneuron degeneration in a pro-inflammatory model of mouse neonatal hypoxia-ischemia.

The pregnancy hormone, human chorionic gonadotropin (hCG) is an immunoregulatory and neurotrophic glycoprotein of potential clinical utility in the neonate at risk for cerebral injury. Despite its well-known role in its ability to modulate the innate immune response during pregnancy, hCG has not been demonstrated to affect the pro-degenerative actions of inflammation in neonatal hypoxia-ischemia (HI). Here we utilize a neonatal mouse model of mild HI combined with intraperitoneal administration of lipopolysaccharide (LPS) to evaluate the neuroprotective actions of hCG in the setting of endotoxin-mediated systemic inflammation. Intraperitoneal treatment of hCG shortly prior to LPS injection significantly decreased tissue loss and cystic degeneration in the hippocampal and cerebral cortex in the term-equivalent neonatal mouse exposed to mild HI. Noting that parvalbumin immunoreactive interneurons have been broadly implicated in neurodevelopmental disorders, it is notable that hCG significantly improved the injury-mediated reduction of these neurons in the cerebral cortex, striatum and hippocampus. The above findings were associated with a decrease in the amount of Iba1 immunoreactive microglia in most of these brain regions. These observations implicate hCG as an agent capable of improving the neurological morbidity associated with peripheral inflammation in the neonate affected by HI. Future preclinical studies should aim at demonstrating added neuroprotective benefit by hCG in the context of therapeutic hypothermia and further exploring the mechanisms responsible for this effect. This research is likely to advance the therapeutic role of gonadotropins as a treatment for neonates with neonatal brain injury.

Mice born preterm develop gait dystonia and reduced cortical parvalbumin immunoreactivity.

Preterm birth leading to cerebral palsy (CP) is the most common cause of childhood dystonia, a movement disorder that is debilitating and often treatment refractory. Dystonia has been typically associated with dysfunction of striatal cholinergic interneurons, but clinical imaging data suggests that cortical injury may best predict dystonia following preterm birth. Furthermore, abnormal sensorimotor cortex inhibition has been found in many studies of non-CP dystonias. To assess the potential for a cortical etiology of dystonia following preterm birth, we developed a new model of preterm birth in mice. Noting that term delivery in mice on a C57BL/6J background is embryonic day 19.1 (E19.1), we induced preterm birth at the limits of pup viability at embryonic day (E) 18.3, equivalent to human 22 weeks gestation. Mice born preterm demonstrate display clinically validated metrics of dystonia during gait (leg adduction amplitude and variability) and also demonstrate reduced parvalbumin immunoreactivity in the sensorimotor cortex, suggesting dysfunction of cortical parvalbumin-positive inhibitory interneurons. Notably, reduced parvalbumin immunoreactivity or changes in parvalbumin-positive neuronal number were not observed in the striatum. These data support the association between cortical dysfunction and dystonia following preterm birth. We propose that our mouse model of preterm birth can be used to study this association and potentially also study other sequelae of extreme prematurity.

Fyn-mediated phosphorylation of Menin disrupts telomere maintenance in stem cells.

Telomeres protect chromosome ends and determine the replication potential of dividing cells. The canonical telomere sequence TTAGGG is synthesized by telomerase holoenzyme, which maintains telomere length in proliferative stem cells. Although the core components of telomerase are well-defined, mechanisms of telomerase regulation are still under investigation. We report a novel role for the Src family kinase Fyn, which disrupts telomere maintenance in stem cells by phosphorylating the scaffold protein Menin. We found that Fyn knockdown prevented telomere erosion in human and mouse stem cells, validating the results with four telomere measurement techniques. We show that Fyn phosphorylates Menin at tyrosine 603 (Y603), which increases Menin's SUMO1 modification, C-terminal stability, and importantly, its association with the telomerase RNA component (TR). Using mass spectrometry, immunoprecipitation, and immunofluorescence experiments we found that SUMO1-Menin decreases TR's association with telomerase subunit Dyskerin, suggesting that Fyn's phosphorylation of Menin induces telomerase subunit mislocalization and may compromise telomerase function at telomeres. Importantly, we find that Fyn inhibition reduces accelerated telomere shortening in human iPSCs harboring mutations for dyskeratosis congenita.

Human endogenous retrovirus K Gag coassembles with HIV-1 Gag and reduces the release efficiency and infectivity of HIV-1.

Human endogenous retroviruses (HERVs), which are remnants of ancestral retroviruses integrated into the human genome, are defective in viral replication. Because activation of HERV-K and coexpression of this virus with HIV-1 have been observed during HIV-1 infection, it is conceivable that HERV-K could affect HIV-1 replication, either by competition or by cooperation, in cells expressing both viruses. In this study, we found that the release efficiency of HIV-1 Gag was 3-fold reduced upon overexpression of HERV-K(CON) Gag. In addition, we observed that in cells expressing Gag proteins of both viruses, HERV-K(CON) Gag colocalized with HIV-1 Gag at the plasma membrane. Furthermore, HERV-K(CON) Gag was found to coassemble with HIV-1 Gag, as demonstrated by (i) processing of HERV-K(CON) Gag by HIV-1 protease in virions, (ii) coimmunoprecipitation of virion-associated HERV-K(CON) Gag with HIV-1 Gag, and (iii) rescue of a late-domain-defective HERV-K(CON) Gag by wild-type (WT) HIV-1 Gag. Myristylation-deficient HERV-K(CON) Gag localized to nuclei, suggesting cryptic nuclear trafficking of HERV-K Gag. Notably, unlike WT HERV-K(CON) Gag, HIV-1 Gag failed to rescue myristylation-deficient HERV-K(CON) Gag to the plasma membrane. Efficient colocalization and coassembly of HIV-1 Gag and HERV-K Gag also required nucleocapsid (NC). These results provide evidence that HIV-1 Gag heteromultimerizes with HERV-K Gag at the plasma membrane, presumably through NC-RNA interaction. Intriguingly, HERV-K Gag overexpression reduced not only HIV-1 release efficiency but also HIV-1 infectivity in a myristylation- and NC-dependent manner. Altogether, these results indicate that Gag proteins of endogenous retroviruses can coassemble with HIV-1 Gag and modulate the late phase of HIV-1 replication.

Expression of human endogenous retrovirus type K (HML-2) is activated by the Tat protein of HIV-1.

Human endogenous retroviruses (HERVs) make up 8% of the human genome. The expression of HERV-K (HML-2), the family of HERVs that most recently entered the genome, is tightly regulated but becomes markedly increased after infection with HIV-1. To better understand the mechanisms involved in this activation, we explored the role of the HIV-1 Tat protein in inducing the expression of these endogenous retroviral genes. Administration of recombinant HIV-1 Tat protein caused a 13-fold increase in HERV-K (HML-2) gag RNA transcripts in Jurkat T cells and a 10-fold increase in primary lymphocytes, and the expression of the HERV-K (HML-2) rec and np9 oncogenes was also markedly increased. This activation was seen especially in lymphocytes and monocytic cells, the natural hosts for HIV-1 infection. Luciferase reporter gene assays demonstrated that the effect of Tat on HERV-K (HML-2) expression occurred at the level of the transcriptional promoter. The transcription factors NF-κB and NF-AT contribute to the Tat-induced activation of the promoter, as shown by chromatin immunoprecipitation assays, mutational analysis of the HERV-K (HML-2) long terminal repeat, and treatments with agents that inhibit NF-κB or NF-AT activation. These studies demonstrate that HIV-1 Tat plays an important role in activating expression of HERV-K (HML-2) in the setting of HIV-1 infection.

Characterization of human endogenous retroviral elements in the blood of HIV-1-infected individuals.

We previously reported finding the RNA of a type K human endogenous retrovirus, HERV-K (HML-2), at high titers in the plasma of HIV-1-infected and cancer patients (R. Contreras-Galindo et al., J. Virol. 82:9329-9236, 2008.). The extent to which the HERV-K (HML-2) proviruses become activated and the nature of their activated viral RNAs remain important questions. Therefore, we amplified and sequenced the full-length RNA of the env gene of the type 1 and 2 HERV-K (HML-2) viruses collected from the plasma of seven HIV-1-infected patients over a period of 1 to 3 years and from five breast cancer patients in order to reconstruct the genetic evolution of these viruses. HERV-K (HML-2) RNA was found in plasma fractions of HIV-1 patients at a density of ∼1.16 g/ml that contained both immature and correctly processed HERV-K (HML-2) proteins and virus-like particles that were recognized by anti-HERV-K (HML-2) antibodies. RNA sequences from novel HERV-K (HML-2) proviruses were discovered, including K111, which is specifically active during HIV-1 infection. Viral RNA arose from complete proviruses and proviruses devoid of a 5' long terminal repeat, suggesting that the expression of HERV-K (HML-2) RNA in these patients may involve sense and antisense transcription. In HIV-1-infected individuals, the HERV-K (HML-2) viral RNA showed evidence of frequent recombination, accumulation of synonymous rather than nonsynonymous mutations, and conserved N-glycosylation sites, suggesting that some of the HERV-K (HML-2) viral RNAs have undergone reverse transcription and are under purifying selection. In contrast, HERV-K (HML-2) RNA sequences found in the blood of breast cancer patients showed no evidence of recombination and exhibited only sporadic viral mutations. This study suggests that HERV-K (HML-2) is active in HIV-1-infected patients, and the resulting RNA message reveals previously undiscovered HERV-K (HML-2) genomic sequences.

[Result of the update of the clinical pathway for hip fracture in the older adult at a university hospital in Madrid]. / Resultado de la actualización de la vía clínica de la fractura de cadera del anciano en un hospital universitario de Madrid.

BACKGROUND AND OBJECTIVES: Orthogeriatric management with clinical pathways (CP) in hip fracture (HF) has been shown to be superior to other models. We studied whether updating the CP, through prioritization of admission and surgery, improvement in the prevention and treatment of delirium, management of anticoagulants and antiplatelet agents and the use of perioperative peripheral nerve block, modifies surgical delay, stay, readmissions, mortality, suffering delirium and functional status at discharge. MATERIAL AND METHOD: A retrospective observational study of unicenter cohorts of 468 patients with HF, 220 from 2016 (old VC) and 248 from 2019 (new VC). The variables are: intervention in the first 48hours, surgical delay (hours), stay (days), stay less than 15 days, delirium, functional loss at discharge (Barthel prefracture scale less Barthel scale at discharge), readmission at one month, and mortality at admission, month and year. RESULTS: Median age: 87.0 [interquartile range 8.0], mostly women (76.7%). Significantly, with the new VC, there was a greater number of patients operated on in the first 48hours (27,7% vs 36,8% p=0.036), less surgical delay (72.5 [47,5-110,5] vs 64.0 [42,0-88,0] p<0.001), shorter stay (10,0 [7,0-13,0] vs 8,0 [6,0-11,0] p<0.001), greater number of discharges in 15 days (78,2% vs 91,5% p<0.001), lower delirium (54,1% vs 43,5% p=0.023). No significant changes in readmissions, functional loss at discharge, mortality at admission, 3 months or year. CONCLUSIONS: Updating the VC brings benefits to the patient (less surgical delay, equal functional status at discharge with fewer days of admission) and benefits in management (lower admission) without modifying mortality.

Benefits of newborn screening and hematopoietic cell transplant in infantile Krabbe disease.

Infantile Krabbe disease (IKD) can be treated with hematopoietic cell transplantation (HCT) if done during the first weeks of life before symptoms develop. To facilitate this, newborn screening (NBS) has been instituted in 8 US states. An application to add IKD to the recommended NBS panel is currently under review. In this report, the outcomes of newborns with IKD diagnosed through NBS and treated with HCT are presented. The unique challenges associated with NBS for this disease are discussed, including opportunities for earlier diagnosis and streamlining treatment referrals. This is a retrospective review of six infants with IKD detected by NBS who were referred for HCT. The timing from diagnosis to HCT was examined, and both HCT and neurodevelopmental outcomes are described. Neurologic testing before HCT revealed evidence of active IKD in all infants. All underwent HCT between 24 and 40 days of age, were successfully engrafted, and are alive 30 to 58 months later (median, 47.5 months). All are gaining developmental milestones albeit at a slower pace than unaffected age-matched peers. Gross motor function is most notably affected. NBS for these patients enabled early access to HCT, the only currently available treatment of infants with IKD. All children are alive and have derived developmental and neurologic benefits from timely HCT. Long-term follow up is ongoing. Optimization of HCT and further development of emerging therapies, all of which must be delivered early in life, are expected to further improve outcomes of infants with IKD.

Centromere defects, chromosome instability, and cGAS-STING activation in systemic sclerosis.

Centromere defects in Systemic Sclerosis (SSc) have remained unexplored despite the fact that many centromere proteins were discovered in patients with SSc. Here we report that lesion skin fibroblasts from SSc patients show marked alterations in centromeric DNA. SSc fibroblasts also show DNA damage, abnormal chromosome segregation, aneuploidy (only in diffuse cutaneous (dcSSc)) and micronuclei (in all types of SSc), some of which lose centromere identity while retaining centromere DNA sequences. Strikingly, we find cytoplasmic "leaking" of centromere proteins in limited cutaneous SSc (lcSSc) fibroblasts. Cytoplasmic centromere proteins co-localize with antigen presenting MHC Class II molecules, which correlate precisely with the presence of anti-centromere antibodies. CENPA expression and micronuclei formation correlate highly with activation of the cGAS-STING/IFN-ß pathway as well as markers of reactive oxygen species (ROS) and fibrosis, ultimately suggesting a link between centromere alterations, chromosome instability, SSc autoimmunity, and fibrosis.