Leveraging network analytics to infer patient syndrome and identify causal genes in rare disease cases.

BACKGROUND: Next-generation sequencing is widely used to identify disease-causing variants in patients with rare genetic disorders. Identifying those variants from whole-genome or exome data can be both scientifically challenging and time consuming. A significant amount of time is spent on variant annotation, and interpretation. Fully or partly automated solutions are therefore needed to streamline and scale this process. RESULTS: We describe Phenotype Driven Ranking (PDR), an algorithm integrated into Ingenuity Variant Analysis, that uses observed patient phenotypes to prioritize diseases and genes in order to expedite causal-variant discovery. Our method is based on a network of phenotype-disease-gene relationships derived from the QIAGEN Knowledge Base, which allows for efficient computational association of phenotypes to implicated diseases, and also enables scoring and ranking. CONCLUSIONS: We have demonstrated the utility and performance of PDR by applying it to a number of clinical rare-disease cases, where the true causal gene was known beforehand. It is also shown that PDR compares favorably to a representative alternative tool.

New Insights into HIV Life Cycle, Th1/Th2 Shift during HIV Infection and Preferential Virus Infection of Th2 Cells: Implications of Early HIV Treatment Initiation and Care.

The theory of immune regulation involves a homeostatic balance between T-helper 1 (Th1) and T-helper 2 (Th2) responses. The Th1 and Th2 theories were introduced in 1986 as a result of studies in mice, whereby T-helper cell subsets were found to direct different immune response pathways. Subsequently, this hypothesis was extended to human immunity, with Th1 cells mediating cellular immunity to fight intracellular pathogens, while Th2 cells mediated humoral immunity to fight extracellular pathogens. Several disease conditions were later found to tilt the balance between Th1 and Th2 immune response pathways, including HIV infection, but the exact mechanism for the shift from Th1 to Th2 cells was poorly understood. This review provides new insights into the molecular biology of HIV, wherein the HIV life cycle is discussed in detail. Insights into the possible mechanism for the Th1 to Th2 shift during HIV infection and the preferential infection of Th2 cells during the late symptomatic stage of HIV disease are also discussed.

Crossroads of Drug Abuse and HIV Infection: Neurotoxicity and CNS Reservoir.

Drug abuse is a common comorbidity in people infected with HIV. HIV-infected individuals who abuse drugs are a key population who frequently experience suboptimal outcomes along the HIV continuum of care. A modest proportion of HIV-infected individuals develop HIV-associated neurocognitive issues, the severity of which further increases with drug abuse. Moreover, the tendency of the virus to go into latency in certain cellular reservoirs again complicates the elimination of HIV and HIV-associated illnesses. Antiretroviral therapy (ART) successfully decreased the overall viral load in infected people, yet it does not effectively eliminate the virus from all latent reservoirs. Although ART increased the life expectancy of infected individuals, it showed inconsistent improvement in CNS functioning, thus decreasing the quality of life. Research efforts have been dedicated to identifying common mechanisms through which HIV and drug abuse lead to neurotoxicity and CNS dysfunction. Therefore, in order to develop an effective treatment regimen to treat neurocognitive and related symptoms in HIV-infected patients, it is crucial to understand the involved mechanisms of neurotoxicity. Eventually, those mechanisms could lead the way to design and develop novel therapeutic strategies addressing both CNS HIV reservoir and illicit drug use by HIV patients.

Up-regulation of HIV-1 transduction in nondividing cells by double-strand DNA break-inducing agents.

Efficient HIV-1 transduction depends on a number of cellular co-factors. Cellular double-strand DNA break (DSB) repair proteins have been proposed, by ourselves and others, to be required for efficient HIV-1 transduction. Expression and/or activity of these DNA repair proteins can be induced by the introduction of DSBs into the host cell genome. HIV-1 transduction was up-regulated by treatment with DSB-inducing agents in both drug-arrested cells and differentiated neuronal cells. The presented data support the hypothesis that DSB repair proteins are involved in the early steps of the retroviral life-cycle.

A role for the histone deacetylase HDAC4 in the life-cycle of HIV-1-based vectors.

HIV-1 integration is mediated by the HIV-1 integrase protein, which joins 3'-ends of viral DNA to host cell DNA. To complete the integration process, HIV-1 DNA has to be joined to host cell DNA also at the 5'-ends. This process is called post-integration repair (PIR). Integration and PIR involve a number of cellular co-factors. These proteins exhibit different degrees of involvement in integration and/or PIR. Some are required for efficient integration or PIR. On the other hand, some reduce the efficiency of integration. Finally, some are involved in integration site selection. We have studied the role of the histone deacetylase HDAC4 in these processes. HDAC4 was demonstrated to play a role in both cellular double-strand DNA break repair and transcriptional regulation. We observed that HDAC4 associates with viral DNA in an integrase-dependent manner. Moreover, infection with HIV-1-based vectors induces foci of the HDAC4 protein. The related histone deacetylases, HDAC2 and HDAC6, failed to associate with viral DNA after infection. These data suggest that HDAC4 accumulates at integration sites. Finally, overexpression studies with HDAC4 mutants suggest that HDAC4 may be required for efficient transduction by HIV-1-based vectors in cells that are deficient in other DNA repair proteins. We conclude that HDAC4 is likely involved in PIR.

Shedding Light on the Role of Extracellular Vesicles in HIV Infection and Wound Healing.

Extracellular vesicles (EVs) play an important role in intercellular communication. They are naturally released from cells into the extracellular environment. Based on their biogenesis, release pathways, size, content, and function, EVs are classified into exosomes, microvesicles (MVs), and apoptotic bodies (ApoBDs). Previous research has documented that EVs, specifically exosomes and MVs, play an important role in HIV infection, either by promoting HIV infection and pathogenesis or by inhibiting HIV-1 to a certain extent. We have also previously reported that EVs (particularly exosomes) from vaginal fluids inhibit HIV at the post-entry step (i.e., reverse transcription, integration). Besides the role that EVs play in HIV, they are also known to regulate the process of wound healing by regulating both the immune and inflammatory responses. It is noted that during the advanced stages of HIV infection, patients are at greater risk of wound-healing and wound-related complications. Despite ongoing research, the data on the actual effects of EVs in HIV infection and wound healing are still premature. This review aimed to update the current knowledge about the roles of EVs in regulating HIV pathogenesis and wound healing. Additionally, we highlighted several avenues of EV involvement in the process of wound healing, including coagulation, inflammation, proliferation, and extracellular matrix remodeling. Understanding the role of EVs in HIV infection and wound healing could significantly contribute to the development of new and potent antiviral therapeutic strategies and approaches to resolve impaired wounds in HIV patients.

CBF-1 Promotes the Establishment and Maintenance of HIV Latency by Recruiting Polycomb Repressive Complexes, PRC1 and PRC2, at HIV LTR.

The C-promoter binding factor-1 (CBF-1) is a potent and specific inhibitor of the human immunodeficiency virus (HIV)-1 LTR promoter. Here, we demonstrate that the knockdown of endogenous CBF-1 in latently infected primary CD4+ T cells, using specific small hairpin RNAs (shRNA), resulted in the reactivation of latent HIV proviruses. Chromatin immunoprecipitation (ChIP) assays using latently infected primary T cells and Jurkat T-cell lines demonstrated that CBF-1 induces the establishment and maintenance of HIV latency by recruiting polycomb group (PcG/PRC) corepressor complexes or polycomb repressive complexes 1 and 2 (PRC1 and PRC2). Knockdown of CBF-1 resulted in the dissociation of PRCs corepressor complexes enhancing the recruitment of RNA polymerase II (RNAP II) at HIV LTR. Knockdown of certain components of PRC1 and PRC2 also led to the reactivation of latent proviruses. Similarly, the treatment of latently infected primary CD4+ T cells with the PRC2/EZH2 inhibitor, 3-deazaneplanocin A (DZNep), led to their reactivation.

Evidence that the Nijmegen breakage syndrome protein, an early sensor of double-strand DNA breaks (DSB), is involved in HIV-1 post-integration repair by recruiting the ataxia telangiectasia-mutated kinase in a process similar to, but distinct from, cellular DSB repair.

Retroviral transduction involves integrase-dependent linkage of viral and host DNA that leaves an intermediate that requires post-integration repair (PIR). We and others proposed that PIR hijacks the host cell double-strand DNA break (DSB) repair pathways. Nevertheless, the geometry of retroviral DNA integration differs considerably from that of DSB repair and so the precise role of host-cell mechanisms in PIR remains unclear. In the current study, we found that the Nijmegen breakage syndrome 1 protein (NBS1), an early sensor of DSBs, associates with HIV-1 DNA, recruits the ataxia telangiectasia-mutated (ATM) kinase, promotes stable retroviral transduction, mediates efficient integration of viral DNA and blocks integrase-dependent apoptosis that can arise from unrepaired viral-host DNA linkages. Moreover, we demonstrate that the ATM kinase, recruited by NBS1, is itself required for efficient retroviral transduction. Surprisingly, recruitment of the ATR kinase, which in the context of DSB requires both NBS1 and ATM, proceeds independently of these two proteins. A model is proposed emphasizing similarities and differences between PIR and DSB repair. Differences between the pathways may eventually allow strategies to block PIR while still allowing DSB repair.

Pentoxifylline suppresses transduction by HIV-1-based vectors.

Pentoxifylline, a caffeine-related compound, was shown to suppress human immunodeficiency virus type 1 (HIV-1) replication. This effect is thought to be mediated by inhibition of tumor necrosis factor-alpha (TNFalpha)-mediated long-terminal repeat (LTR)-driven expression. We now demonstrate that pentoxifylline efficiently inhibits transduction by HIV-1-based vectors. This latter effect is independent of LTR-driven expression, and correlates with a reduced efficiency of the completion of the integration process in infected cells. Finally, the effect of pentoxifylline is dramatically reduced in cells expressing a dominant negative ATR protein, and in primary human cells that exhibit low level of ATR activity, suggesting that the effect of pentoxifylline on HIV-1 transduction and replication is at least partly mediated by suppression of the ATR kinase.

Management of hyperglycemia in the neurosurgery patient.

Hyperglycemia is associated with adverse outcomes in patients who are candidates for or underwent neurosurgical procedures. Specific concerns and settings that relate to these patients are preoperative glycemic control, intraoperative control, management in the neurological intensive care unit (NICU), and postoperative control. In each of these settings, physicians have to ensure appropriate glycemic control to prevent or minimize adverse events. The glycemic control is usually managed by a neurohospitalist in co-management with the neurosurgery team pre- and post-operatively, and by the neurocritical care team in the setting of NICU. In this review article, we outline current standards of care for neurosurgery patients with diabetes mellitus and/or and hyperglycemia and discuss results of most recent clinical trials. We highlight specific concerns with regards to glycemic controls in these patients including enteral tube feeding and parenteral nutrition, the issues of the transition to the outpatient care, and management of steroid-induced hyperglycemia. We also note lack of evidence in some important areas, and the need for more research addressing these gaps. Where possible, we provide suggestions how to manage these patients when there is no underlying guideline.

Comportamiento y gravedad del trauma maxilofacial en urgencia en el Hospital Universitario "General Calixto García" / Characteristics and Severity of Maxillofacial Trauma in the Emergency Room at General Calixto García University Hospital

Introducción: La región maxilofacial es vulnerable al trauma. Se utilizan herramientas para evaluar la gravedad del trauma maxilofacial. Objetivo: Caracterizar el comportamiento y gravedad del trauma maxilofacial en los pacientes atendidos en el servicio de urgencias de cirugía maxilofacial del Hospital Universitario "General Calixto García". Métodos: Se realizó un estudio observacional descriptivo transversal en pacientes que acudieron al servicio de urgencias de cirugía maxilofacial del Hospital Universitario "General Calixto García", desde septiembre de 2018 hasta marzo de 2021. El universo fue de 57 pacientes. Se aplicó la escala de severidad de lesiones faciales. Variables analizadas: edad, sexo, etiología, diagnósticos, exámenes complementarios, gravedad del trauma maxilofacial, procederes terapéuticos inmediatos. Resultados: Prevaleció la fractura mandibular como diagnóstico (66,7 por ciento). La etiología más frecuente fue la violencia interpersonal (29,8 por ciento). Predominó la gravedad leve del trauma maxilofacial (71,9 por ciento). La extracción de cuerpos extraños de la vía aérea, tracción lingual, inserción de cánula orofaríngea, intubación orotraqueal (5,3 por ciento), y sutura (64,9 por ciento), fueron los procederes terapéuticos inmediatos más utilizados. Conclusiones: Prevalecen los pacientes del sexo masculino y del grupo de edad de 19 a 30 años. Predomina la fractura mandibular como diagnóstico. Los exámenes complementarios más utilizados son, la tomografía computarizada, el hemograma completo y el coagulograma. Impera la violencia interpersonal como etiología. Prevalece la gravedad leve del trauma maxilofacial. Los procederes terapéuticos inmediatos más empleados son, extracción de cuerpos extraños de la vía aérea, tracción lingual, inserción de cánula orofaríngea, intubación orotraqueal y sutura(AU)

Introduction: The maxillofacial region is vulnerable to trauma. Tools are used to assess the severity of maxillofacial trauma. Objective: To characterize maxillofacial trauma and its severity in patients cared for at the maxillofacial surgery emergency department of General Calixto García University Hospital. Methods: A cross-sectional, descriptive and observational study was carried out in patients attending the maxillofacial surgery emergency department at General Calixto García University Hospital, from September 2018 to March 2021. The study universe was 57 patients. The facial injury severity scale was applied. The analyzed variables were age, sex, etiology, diagnoses, complementary examinations, severity of maxillofacial trauma, immediate therapeutic procedures. Results: Mandibular fracture prevailed as a diagnosis (66.7 percent). The most frequent etiology was interpersonal violence (29.8 percent). Mild severity of maxillofacial trauma predominated (71.9 percent). Extraction of foreign bodies from the airway, tongue traction, insertion of oropharyngeal cannula, orotracheal intubation (5.3 percent) and suturing (64.9 percent) were the most frequently used immediate therapeutic procedures. Conclusions: There is a prevalence of male patients and the age group 19 to 30 years. Mandibular fracture predominates as a diagnosis. The most frequently used complementary tests are computed tomography, complete blood count and coagulogram. Interpersonal violence prevails as an etiology. Mild severity of maxillofacial trauma prevails. The most commonly used immediate therapeutic procedures are extraction of foreign bodies from the airway, tongue traction, insertion of oropharyngeal cannula, orotracheal intubation and suturing(AU)

Human vaginal fluid contains exosomes that have an inhibitory effect on an early step of the HIV-1 life cycle.

OBJECTIVE: Vaginal transmission is crucial to the spread of HIV-1 around the world. It is not yet clear what type (s) of innate defenses against HIV-1 infection are present in the vagina. Here, we aimed to determine whether human vaginal fluid contains exosomes that may possess anti-HIV-1 activity. METHODS: The exosomal fraction was isolated from samples of vaginal fluids. The presence of exosomes was confirmed by flow cytometry and western blotting. The newly discovered exosomes were tested for their ability to block early steps of HIV-1 infection in vitro using established cell culture systems and real time PCR-based methods. RESULTS: Vaginal fluid contains exosomes expressing CD9, CD63, and CD81 exosomal markers. The exosomal fraction of the fluid-reduced transmission of HIV-1 vectors by 60%, the efficiency of reverse transcription step by 58.4%, and the efficiency of integration by 47%. Exosomes had no effect on the entry of HIV-1 vectors. CONCLUSION: Human vaginal fluid exosomes are newly discovered female innate defenses that may protect women against HIV-1 infection.

Herramientas pronósticas de la gravedad del trauma maxilofacial en la atención de urgencia / Prognostic Tools for Maxillofacial Trauma Severity in Emergency Care

RESUMEN Introducción: La utilización de herramientas pronósticas que permitan valorar la gravedad del trauma, la supervivencia y mortalidad de los lesionados, permite la estratificación de los pacientes y la toma de decisiones acertadas para el tratamiento más efectivo en el área hospitalaria. Objetivo: Realizar una revisión bibliográfica sobre las herramientas pronósticas más utilizadas en la atención de urgencia para detectar la gravedad del trauma maxilofacial. Métodos: Se realizó una revisión bibliográfica desde octubre de 2018 hasta enero de 2019. Fueron examinados libros y artículos publicados en revistas de impacto, en idioma español, inglés y portugués, con atención prioritaria a aquellos que tuvieran cinco años o menos. Se consultaron las bases de datos MEDLINE, EBSCO, PubMed, SciELO, ClinicalKey, utilizando los descriptores: "Trauma Severity Indices", "Maxillofacial Injuries", "Traumatology", "Indexes". Se recuperaron dos libros y 50 artículos, aunque la revisión se circunscribió solamente a los 33 que mejor describían los elementos estudiados. Análisis e integración de la información: El puntaje Cooter David, el de gravedad de lesiones maxilofaciales, la escala de severidad de lesiones faciales, la de severidad de fracturas faciales y el modelo ZS, son los principales instrumentos utilizados en el pronóstico de gravedad existentes de la región facial. Conclusiones: Constan en la literatura escalas, índices y puntajes para pronosticar la gravedad del trauma maxilofacial en la urgencia, cada una exhibe ventajas que las convierte en una herramienta coadyuvante de la atención del trauma; sin embargo, poseen también desventajas que complejizan su aplicación en el medio hospitalario. Entre las herramientas más utilizadas está la escala de severidad de lesiones faciales; fácil de aplicar en los servicios de urgencia, pero con deficiencias a la hora de tratar los componentes de tejido blando y óseo. En Cuba hay escasas evidencias de la utilización de estos instrumentos en la especialidad de cirugía maxilofacial(AU)

ABSTRACT Introduction: The usage of prognostic tools that allow assessing trauma severity, as well as injury survival and mortality, allows patient stratification and making right decisions for the most effective treatment in the hospital area. Objective: To carry out a bibliographic review about the prognostic tools most widely used in emergency care to identify maxillofacial trauma severity. Methods: A bibliographic review was carried out from October 2018 to January 2019. We examined books and articles published in high-impact magazines, in Spanish, English and Portuguese, with priority attention on those five years old or less. We consulted the MEDLINE, EBSCO, PubMed, SciELO, Clinical Key databases using the descriptors Trauma Severity Indices, Maxillofacial Injuries, Traumatology, and Indexes. Two books and 50 articles were retrieved, although the review was limited to only 33 which described best the elements studied. Information analysis and integration: The Cooter-David scoring system, the maxillofacial injury severity score, the facial injury severity scale, the facial fracture severity scale, and the ZS model are the main instruments used for existing severity forecast in the facial region. Conclusions: There literature includes scales, indexes and scoring systems for predicting the maxillofacial trauma severity in the emergency department, each one showing advantages that make them an adjunct tool for trauma care; however, they also have disadvantages that complicate their application in the hospital setting. The facial injury severity scale is among the most widely used tools, as far as it is easy to apply in the emergency services, but with deficiencies for treating soft tissue and bone components. In Cuba, there is little evidence about the use of these instruments in the specialty of maxillofacial surgery(AU)

Manifestaciones bucales de la diabetes mellitus tipo 2 en el adulto mayor / Oral manifestations of type 2 diabetes mellitus in the elderly

Introducción: La diabetes mellitus tipo 2 favorece la aparición de enfermedades bucales en el adulto mayor. Objetivos: Identificar las alteraciones bucales y factores de riesgo más frecuentes en pacientes con diabetes mellitus tipo 2. Métodos: Se realizó un estudio observacional, descriptivo y transversal. El universo lo conformaron 52 pacientes pertenecientes al consultorio # 13 del Policlínico Héroes del Corynthia, atendidos desde 2016 a 2017 en la Clínica Estomatológica Docente H y 21, municipio Plaza de la Revolución. Las variables analizadas fueron: edad, sexo, estado de salud bucal, factores de riesgo y manifestaciones bucales. Resultados: 55,7 por ciento de los adultos mayores pertenecieron al grupo de 60-69 años y el 59,4 por ciento del total de los pacientes involucrados en el estudio fueron mujeres. El 44,2 por ciento presentó un estado de salud bucal medianamente favorable. Por caries dental fueron afectados 34 pacientes, 31 por periodontitis y 25 por gingivitis. La obesidad se evidenció en el 59,6 por ciento de los pacientes. Conclusiones: El factor de riesgo predominante fue la obesidad. Las alteraciones bucales más observadas fueron la caries dental, la periodontitis y la gingivitis(AU)

Introduction: Type 2 diabetes mellitus fosters the appearance of oral diseases in the elderly. Objectives: Identify the most common oral alterations and risk factors among patients with type 2 diabetes mellitus. Methods: A cross-sectional observational descriptive study was conducted. The study universe was 52 patients from Consultation Office No. 13 of Héroes del Corynthia polyclinic cared for from 2016 to 2017 at H and 21st University Dental Clinic in the municipality of Plaza de la Revolución. The variables analyzed were age, sex, oral health status, risk factors and oral manifestations. Results: 55.7 percent of the elderly patients were in the 60-69 age group, and 59.4 percent of the total patients involved in the study were women. 44.2 percent had a fairly favorable oral health status. 34 had dental caries, 31 periodontitis and 25 gingivitis. 59.6 percent were obese. Conclusions: Obesity was the prevailing risk factor. The oral alterations most commonly found were dental caries, periodontitis and gingivitis(AU)

Manifestaciones bucales de la diabetes mellitus en el adulto mayor / Oral manifestations of diabetes mellitus in elder people

RESUMEN A lo largo de la historia, el hombre siempre ha estado preocupado por el envejecimiento, siendo en la actualidad un fenómeno mundial sin precedentes en la historia de la humanidad. Entre las enfermedades crónicas características de la ancianidad se encuentra la diabetes mellitus, siendo esta una enfermedad compleja endocrino metabólica, de curso crónico, que puede favorecer la aparición de enfermedades bucales. El objetivo fue caracterizar a la población adulta mayor con diabetes mellitas e identificar las principales manifestaciones bucales provocadas por la enfermedad. Se reunió información a través de los buscadores de información y plataformas científicas: Pubmed, Scielo, Springer, ClinicalKey. Se consultaron un total de 36 artículos. Como resultados más relevantes se constató que el envejecimiento es un proceso que con el tiempo le ocurre a todo ser vivo a consecuencia de la interacción de la genética del individuo con su medio, y que la caries dental, la peridontitis y la gingivitis son las manifestaciones bucales más frecuentes de la diabetes mellitus. Existe una relación importante entre la diabetes mellitus y la aparición de las manifestaciones bucales (AU).

ABSTRACT Through the history, the man has been always worried about ageing; currently it is worldwide phenomena without precedents in the history of the human kind. Diabetes mellitus is among the chronic diseases that are characteristic of the elder age, being this an endocrine-metabolic complex disease, of chronic curse, that may contribute to the emergence of oral diseases. The objective was characterizing elder population with Diabetes mellitus and to identify the main oral manifestations caused for that disease. The information was gathered using information searchers and scientific platforms: Pubmed, Scielo, Springer, ClinicalKey. 36 articles were consulted. As the most relevant results were stated that ageing is a process occurring to any living creature as a consequence of the interaction of the individual's genetic and his environment, and that dental caries, periodontitis and gingivitis are the most frequent oral manifestations of the Diabetes mellitus. There is an important relation between Diabetes mellitus and the emergence of oral manifestations (AU).

Stem cells and aging: a chicken-or-the-egg issue?

Aging is a process that involves all organs and tissues of the human organism. Cells and tissues are impacted by aging in differing degrees, depending on their regenerative potential and sensitivity to outside stimuli. In this review, we discuss the potential role of adult stem cells in the aging process, and the new results that support the role of stem cells in the aging process. Finally, we discuss new evidence from progeroid syndromes that supports the stem cell hypothesis of aging.

Proliferating cell nuclear antigen is required for loading of the SMCX/KMD5C histone demethylase onto chromatin.

BACKGROUND: Histone methylation is regulated by a large number of histone methyltransferases and demethylases. The recently discovered SMCX/KMD5C demethylase has been shown to remove methyl residues from lysine 4 of histone H3 (H3K4), and constitutes an important component of the regulatory element-1-silencing transcription factor (REST) protein complex. However, little is known about the cellular mechanisms that control SMCX activity and intracellular trafficking. RESULTS: In this study, we found that small interfering RNA-mediated knockdown of proliferating cell nuclear antigen (PCNA) resulted in the reduction of the chromatin-bound SMCX fraction. We identified a PCNA-interaction protein motif (PIP box) in the SMCX protein. Using site-directed mutagenesis, we found that the amino acids of the SMCX PIP box are involved in the association of SMCX with PCNA and its interaction with chromatin. CONCLUSIONS: Our data indicate that the intracellular trafficking of SMCX is controlled by its association with PCNA.

HIV-1 integration site preferences in pluripotent cells.

HIV-1-based vectors are widely used in gene therapy. In somatic cells, these vectors mainly integrate within genes. However, no distinct integration site preferences have been observed with regard to large chromosomal regions. The recent emergence of induced pluripotent stem (iPS) cells, similar to embryonic stem (ES) cells, has raised questions about where integration occurs in these cells. In this work we investigated the integration site preferences of HIV-1-based vectors in a pluripotent, ES-like cell line. We show that approximately 30% of the integrations occur in the vicinity of telomeres. We have analyzed integration sites in various somatic cells, as reported by us and other groups, and observed that this integration pattern is unique to the analyzed pluripotent cell line. We conclude that pluripotent cells may contain distinct cellular cofactors that participate in integration targeting and that are not present in somatic cells.

Pyruvate kinase expression (PKM1 and PKM2) in cancer-associated fibroblasts drives stromal nutrient production and tumor growth.

We have previously demonstrated that enhanced aerobic glycolysis and/or autophagy in the tumor stroma supports epithelial cancer cell growth and aggressive behavior, via the secretion of high-energy metabolites. These nutrients include lactate and ketones, as well as chemical building blocks, such as amino acids (glutamine) and nucleotides. Lactate and ketones serve as fuel for cancer cell oxidative metabolism, and building blocks sustain the anabolic needs of rapidly proliferating cancer cells. We have termed these novel concepts the "Reverse Warburg Effect," and the "Autophagic Tumor Stroma Model of Cancer Metabolism." We have also identified a loss of stromal caveolin-1 (Cav-1) as a marker of stromal glycolysis and autophagy. The aim of the current study was to provide genetic evidence that enhanced glycolysis in stromal cells favors tumorigenesis. To this end, normal human fibroblasts were genetically-engineered to express the two isoforms of pyruvate kinase M (PKM1 and PKM2), a key enzyme in the glycolytic pathway. In a xenograft model, fibroblasts expressing PKM1 or PKM2 greatly promoted the growth of co-injected MDA-MB-231 breast cancer cells, without an increase in tumor angiogenesis. Interestingly, PKM1 and PKM2 promoted tumorigenesis by different mechanism(s). Expression of PKM1 enhanced the glycolytic power of stromal cells, with increased output of lactate. Analysis of tumor xenografts demonstrated that PKM1 fibroblasts greatly induced tumor inflammation, as judged by CD45 staining. In contrast, PKM2 did not lead to lactate accumulation, but triggered a "pseudo-starvation" response in stromal cells, with induction of an NFκB-dependent autophagic program, and increased output of the ketone body 3-hydroxy-buryrate. Strikingly, in situ evaluation of Complex IV activity in the tumor xenografts demonstrated that stromal PKM2 expression drives mitochondrial respiration specifically in tumor cells. Finally, immuno-histochemistry analysis of human breast cancer samples lacking stromal Cav-1 revealed PKM1 and PKM2 expression in the tumor stroma. Thus, our data indicate that a subset of human breast cancer patients with a loss of stromal Cav-1 show profound metabolic changes in the tumor microenvironment. As such, this subgroup of patients may benefit therapeutically from potent inhibitors targeting glycolysis, autophagy and/or mitochondrial activity (such as metformin).