There were significantly higher numbers of AIDS and non-AIDS events in the HBcAb+/HBsAb+/−-positive subjects than in the HBV-negative patients ; additionally, HIV viremia undetectability was achieved a significantly longer time after cART was begun in the former than in the latter population . Cox multivariable analysis confirmed that after starting cART, an HBcAb+/HBsAb+/−-positive status is a risk factor for a lower odds of achieving virological success and a higher risk of experiencing virological rebound . HBcAb-positive status resulted in a delay in achieving HIV < 50 copies/mL and the appearance of viral rebound in course of cART, hence it is related to a poor control of HIV infection in a population of coinfected patients.The morbidity and mortality rates of human immunodeficiency virus (HIV)-hepatitis B virus (HBV) coinfection are higher than that of either infection alone. Outcomes and the virological response to antiretrovirals  were explored in HIV/HBV subjects in a cohort of Italian patients treated with cART. A single-center retrospective analysis of patients enrolled from January 2007 to June 2018 was conducted by grouping patients by HBV status and recording baseline viro-immunological features, the history of virological failure, the efficacy of cART in achieving HIV viral undetectability, viral blip detection and viral rebound on follow up. Among 231 enrolled patients, 10 (4.3%) were HBV surface (s) antigen (HBsAg)-positive, 85 (36.8%) were positive for antibodies to HBV c antigen (HBcAb) and with or without antibodies to HBV s antigen (HBsAb), and 136 were (58.9%) HBV-negative. At baseline, HBcAb/HBsAb HBsAg-positive cART-treated patients demonstrate impaired CD4 recovery and accelerated evolution towards AIDS3 in addition to increased HBV replication, liver disease progression, hepatocellular carcinoma (HCC) prevalence and liver-related mortality5.Due to the introduction of combined antiretroviral therapy (cART), and in particular the introduction of drugs with activity against hepatitis B virus (HBV), such as Lamivudine (LAM), Tenofovir (TDF) and other nucleoside/nucleotide analogues (NA), the ability to control HBV infection has strongly increased in HIV-coinfected patients. However, compared to HIV mono-infected patients, HIV/HBV coinfected patients reach immune-virologic control with more difficulty and have a higher rate of acquired immunodeficiency syndrome (AIDS) events and end-stage liver disease (ESLD) despite the introduction of cART6 recommended the use of anti-HBV active drugs (NAs) containing cART in HIV-positive patients and strongly suggested avoiding the interruption of this type of treatment. Conversely, no suggestions were provided regarding the management of HIV-positive patients with HBV-resolved infections . Resolved HBV infection is known to be associated with HBV reactivation in immunocompromised patients subsequent to transplantation or chemotherapy for solid or hematological neoplasia7. As a component of resolved infection, occult hepatitis B Infection (OBI), which is defined as the absence of the HBs antigen (HBsAg) in the presence of intrahepatic or plasma HBV replication, is also known to be a risk factor for the evolution of HBV infection in cirrhosis, ESLD and HCC in immunocompromised patients8. Very few data are available regarding the influence of resolved HBV infection and OBI on HIV infection control10, especially in people with low-prevalence HBV infection.Based on these data, the European Clinical Practice Guidelines for the management of HBV infectionThe goal of this study was to explore whether having a resolved HBV infection affects overall health outcomes by comparing a group of cART-treated, HBV/HIV-coinfected Italian subjects to a group of HIV mono-infected patients. Clinical, virological and immunological data of patients seen from January 2007 until July 2018 at the Infectious Diseases Unit of the Policlinico Tor Vergata were retrospectively evaluated.An observational retrospective study of 671 HIV-positive patients collected from January 2007 to July 2018 at the Infectious Diseases Unit of the Policlinico Tor Vergata in Rome, Italy was conducted. Specifically, for this study, a database was built that included all patients’ data at baseline, at the start of cART treatment, and at follow-up visits. In particular, the following data were collected for all of the patients: hematochemical data and HBV and HCV serology at baseline, HIV-RNA viral load, CD4+ cell count at baseline and pre-cART, time since the HIV infection diagnosis and the start time of cART treatment. Data on flares in transaminases (defined as an increase in aspartate aminotransferase (AST) or alanine aminotransferase (ALT) > 50 IU/L), HIV viral blips , virological rebound  and time required to achieve viral undetectability  were collected during follow-up examinations. Moreover, data regarding the antiviral treatment for chronic hepatitis B (CHB)  were also gathered. When available, HBV-DNA viral load data were collected.No specific ethics committee’s consent was required due to the retrospective nature of the study, which is based on information available from existing clinical documentation [Determination of the Italian Drug Agency (AIFA) of 20 March 2008]. With regard for privacy, all personal information was treated in a confidential manner, and all clinical data were anonymously analyzed.As shown in Fig. +/HBsAb+/− status on time required to achieve an HIV viral load < 50 copies/mL after the initiation of cART and the onset of VB or VR after HIV VS. Subsequently, the effects of HBV antigen/antibody profiles  on overall mortality and the appearance of AIDS-related events were also evaluated.The primary endpoint of this study was to investigate the influence of HBcAbFurthermore, non-AIDS events, including hypertension, diabetes and lipidic disorders, bone disorders , renal impairment and non-AIDS-related neoplastic pathologies, were investigated.HBV serological markers were measured using immune-enzymatic assays . Plasma HBV-DNA was identified using real-time polymerase chain reaction (lower limit of quantification: 20 IU/ml) . Plasma HIV-RNA levels were measured using a commercial test, with 20 copies/ml of HIV-RNA defined as the lower limit of quantification .All statistical analyzes were conducted using STATA 14.2 .+/HBsAb+/−-positive and HBV-negative patients was performed with the Kruskal-Wallis test for continuous variables and with the Chi-squared test or Fisher’s exact test, when appropriate, for categorical variables.The study population is described using proportions and percentages for categorical values and median measurements and interquartile ranges (IQRs) for continuous values. The comparison between HBcAb+/HBsAb+/−-positive and HBV-negative groups of patients.Kaplan-Meier curves were used to estimate the time required to achieve and the probability of achieving VS after the start of cART and the probabilities of experiencing VB and VR in the HBcAbCox regression analysis was performed to evaluate the association between HBcAb and the risk of achieving VS or experiencing VR after controlling for other potential confounding factors under the assumption of proportionality of the hazards. The following variables were considered potential confounders: age, AST levels, hepatitis C virus (HCV) coinfection, pre-cART plasma HIV-RNA, pre-cART CD4 count, anti-HBV treatment, risk factor for HIV acquisition, calendar year, and type of cART.In Table One hundred seventy-one patients (74.3%) achieved HIV viremia < 50 copies/mL within a median of 5 months (IQR 3–6.5). The large majority (92.6%) of patients were treated with cART-containing anti-HBV agents: 38 (16.4%) with LAM and 176 (76.2%) with TDF or TAF. Overall, during the observation period, AIDS-related events were reported in 64 (27.7%) patients, and 90 (38.9%) developed non-AIDS-related events. HIV VR occurred in 34 (14.8%) subjects, and 101 (44.1%) showed occasional HIV VB during the follow-up period.Pneumocystis jirovecii pneumonia [PJP]), and another 3 patients (30%) developed a non-AIDS-related event (2 cases of cardiovascular disease and 1 of diabetes complications) (data not shown).Ten (4.3%) out of 231 patients were HBsAg-positive; these patients had a median pre-cART CD4 cell count of 193 cell/mmc (IQR 98-373) and an HIV viral load of 87.100 copies/mL (IQR 31.100–143.000). All had detectable plasma HBV-DNA  and were taking cART: 2 (20%) as a regimen containing LAM and 8 (80%) as a regimen containing TDF or TAF. Of these 10 patients, 2 (20%) died because of HCC, 1 (10%) had HCC treated with chemoembolization, 2 (20%) developed an AIDS-related event  and more likely to have a history of intravenous drug use . Compared to HBV-negative patients, HBcAb+/HBsAb+/−-positive patients had more frequent episodes of transaminase flares , significantly lower CD4+ cell counts at baseline , and lower pre-cART CD4+ cell counts .A comparison between HBV-negative and HBcAb+/HBsAb+/−-positive patients had an NA in their cART composition), HBV-negative patients achieved HIV-RNA undetectability in a significantly shorter time than was found in HBcAb+/HBsAb+/−-positive subjects . A significantly higher number of AIDS-related and non-AIDS-related events were detected in HBcAb+/HBsAb+/−-positive subjects than in HBV-negative patients . Among HBcAb+/HBsAb+/− patients, the following AIDS- and non-AIDS-related events were documented: PJP (16 cases), Kaposi Sarcoma (KS) (7 cases), esophageal candidiasis (4 cases), wasting syndrome (4 cases), PJP plus esophageal candidiasis (1 case), non-AIDS tumors (12 cases), lipidic disorders (19 cases), osteoporosis (6 cases), diabetes (5 cases) and more than one non-AIDS disease (7 cases) (data not shown). HBV-negative patients had the following AIDS and non-AIDS pathologies: PJP (14 cases), KS (2 cases), cerebral lymphoma (1 case), esophageal candidiasis (7 cases), more than one of the above-listed AIDS events (2 cases), non-AIDS tumors (7 cases), lipid disorders (13 cases), osteoporosis (2 cases), hypertension (9 cases) or more than one of the previously mentioned non-AIDS comorbidities (8 cases) (data not shown).Once cART was initiated (89% of HBcAb+/HBsAb+/−-positive and HBV-negative patients [p = 0.56]), 62 (74.7%) of the HBcAb+/HBsAb+/−-positive and 30 (22.1%) of the HBV-negative subjects developed HIV VB (p < 0.0001). Moreover, HBcAb+/HBsAb+/− patients were more prone to VR onset . Virological control of HIV during follow-up was poorer in the HBcAb+/HBsAb+/− group. Overall, at the end of the follow-up period, 8 out of 221 patients had died. Of these, six (7.1%) were HBcAb-positive, and only two (1.5%) were HBV-negative (p 0.006).At the end of the follow-up period  . Patients who experienced VR had a higher number of blips (median number of VB 2 [IQR 1–3]) compared to those who did not experience VR (median number of VB 0 [IQR 0–1]) (p < 0.001 Mann-Whitney test) (data not shown).Finally, concerning VR, in Fig. Therefore, Kaplan Meier estimates showed that antiHBc-positive patients exhibited less virological control from the beginning of cART, including a delayed VS and subsequently more frequent occurrence of VB and VR.Table In our cohort of HIV-positive patients who started cART, the presence of serological markers of a past HBV infection was significantly correlated with a delay in achieving HIV viremia undetectability and the appearance of HIV VR after the start of cART . Moreover, significantly higher numbers of AIDS and non-AIDS-related events were found in the group of HIV/HBcAb-positive patients than in HIV mono-infected individuals.12.In our population, 41% of the 231 patients included in this study had an ongoing or resolved HBV infection. Slightly lower rates of HBV-resolved infection or CHB were found in two American cohorts, which reported values of 23% and 35% in HIV seroconverters and HIV patients on cART, respectively13. Very little is known about the influence of resolved HBV infection on the course of HIV infection. From the results of a longitudinal study performed in a large cohort of HIV-negative and -positive patients7, the authors concluded that the isolated presence of HBcAb was a sign of an impaired immune response in patients with an HIV infection.CHB in HIV-positive subjects has been demonstrated to worsen the prognosis of both infections, and a higher risk of mortality was recently observed in patients coinfected with CHB and an HBV DNA viral load > 2000 UI/ml+/HBsAb+/−-positive subjects presented flares of transaminases more frequently than was found in HBV-negative patients , suggesting the potentially transient reactivation of HBV. Hepatitis flares have been widely documented in HBcAb-positive patients during immunosuppressive therapy7, whereas the association of liver diseases with signs of resolved HBV infection in coinfected HIV people is difficult to ascertain as few studies have assessed the association between liver damage and both isolated anti-HBc patterns and occult HBV infections14. In the Veterans Aging Cohort Study15, the presence of HBcAb was associated with more advanced hepatic fibrosis in HIV-HCV coinfected patients, while Morsica and his colleagues9 showed that 21% of HBcAb-positive HIV-coinfected patients in the Italian national cohort (ICONA) presented detectable HBV-DNA associated with an increase in transaminase levels.In our study cohort, HBcAb12, who observed that the risk of AIDS and death was high in both CHB and resolved HBV subjects from a cohort of 2536 HIV-positive cART recipients. Similarly, HBV coinfection negatively impacted patient survival in a group of HIV seroconverters, among whom the rates of AIDS and death were significantly higher in CHB patients and increased, although not significantly, in patients with resolved HBV infections11.An increased number of AIDS and non-AIDS events were documented in the follow-up of our study cohort, which is associated with ongoing or resolved HBV infections. A similar result was reported by Chun HM and his collaborators16. HIV exerts direct activity on hepatocytes and Kupffer cells, thereby contributing to liver inflammation. Moreover, the rate of microbial translocation was higher in HIV/HBV-coinfected individuals than in HBV mono-infected individuals17. Microbial translocation has been associated with ongoing immune dysfunction and contributes to the persistence of chronic immune activation and inflammation, which have been associated with the development of comorbidities in HIV-infected persons, even those receiving effective cART19.A series of potential factors have been shown to form the basis of the HBV and HIV interaction and to result in the acceleration of both infection courses+/− patients than in HIV mono-infected subjects in a multivariate analysis, and the status of the resolved HBV coinfection was strongly correlated with HIV VB onset in HIV monoinfected people .In our study cohort, virological control was worse in HIV/HBcAb/HBsAb20, and some authors have found a correlation between VB and subsequent VR23. In our study, we found that the median VB was higher in patients with VR than in those without VR. The origin of VB remains uncertain, but an increased HIV replication rate has been correlated with the presence of other infections and with certain vaccinations25. A number of viral coinfections, when present in HIV-positive subjects, seem to contribute to unfavorable outcomes. Cytomegalovirus coinfection was associated with HIV disease progression27, and hepatitis C virus is a leading cause of non-AIDS-related mortality among HIV-coinfected patients28.The appearance of VB is commonly described in cART-treated HIV patients29, and is related to a poor immune status8. Therefore, it is possible to argue that occasional, although limited, HBV replications may also contribute to the appearance of HIV viremia during the course of cART.HBV DNA is less rarely detected among patients with HIV and those with isolated anti-HBc (OBI patients), with its rate reported to vary from 0.6% to 89% in several studiesBefore any conclusions can be drawn, some of the limitations of our study need to be discussed. First, the lack of HBV-DNA data in our population was a major limitation. Currently, no shared indications are used in the monitoring of HBV viremia in coinfected patients with resolved HBV infections, but it may be interesting to ascertain whether poor control of HIV viremia during cART is correlated with the simultaneous presence of HBV-DNA in patients with resolved HBV infections. Second, all of the evaluated patients were included from a single institution, and this might reduce the generalizability of our conclusions. Third, the retrospective nature of this study limits the power of the obtained results.In conclusion, during the course of cART, an HBcAb-positive status was a risk factor delayed achievement of HIV < 50 copies/mL and the appearance of VR in our cohort of HIV/HBV-coinfected patients. Further studies are needed to assess how HBV-resolved infections exert a harmful effect on HIV and how they might reduce HIV replication control.


In vivo regulatory functions have evolved from R-loops, including regulation of gene expression and telomere lengthening. However, they also exist as a form of stress, particularly when replication forks collide with the transcription machinery. New methodologies and models are being developed to delineate the biology of R-loops, including those related to cell stress-based diseases like cancer. As accumulation of R-loops is associated with disease, targeting molecular pathways that regulate their formation or removal could provide new avenues for therapeutic intervention. This review covers recent understandings of the molecular basis for R-loop formation, removal, and biological outcomes in the context of cellular stress.Exposure of genomic, single-stranded DNA (ssDNA) during transcription and replication creates opportunities for the formation of inappropriate secondary structures. Cells manage this exposure by using topoisomerases and helicases to reduce the inherent topological stress that arises from unwinding the double helix and by coating ssDNA with protective protein complexes. Interestingly, specific DNA-RNA hybrids, known as R-loops, form during transcription and exist in homeostasis throughout the genomes of prokaryotes and eukaryotes. These hybrids nucleate from guanine rich clusters in the template strand and extend across GC rich spans of transcribed genes.  A variety of topological, structural and hybridization events occur during DNA replication and gene transcription. Unwinding of the DNA double helix provides access for polymerase to a template strand, and creates torsional stress that can manifest anomalous formation of “non-traditional” moieties. One such structure, known as an R-loop , 2, occuR-loops canonically nucleate from guanine-rich clusters (G-clusters) during gene transcription , 4, and The build-up of R-loops is counteracted through a variety of mechanisms that maintain transcription bubble integrity, including nuclease activity and resolution of topological stress  speed and potentiates fork stalling, factors that contribute to R-loop nucleation. Furthermore, recruitment of the repair machinery to sites of DNA damage occurs through MOF-mediated ubiquitination , while aCollectively, the cooperative activities of transcription bubble maintenance and mRNA processing machinery resolve and prevent R-loop accumulation. Each individual component contributes to the homeostasis of R-loops, and their respective regulation determines the local prevalence of these hybrid structures throughout the genome.in vitro and in vivo. Various plasmid-based systems have been created to study the regulation of R-loop formation and removal. Known R-loop sequences are inserted into plasmids in order to examine the effect of these structures on replication or transcription in vitro.Since being first described in 1976 , severalin vitro. The R-loop structure migrates more slowly in agarose gel electrophoresis and can be visualized with ethidium bromide (EtBr) after in vitro transcription [in vitro transcription step increases sensitivity of the assay. Moreover, it eliminates signal from dsDNA that appears when using a general nucleic acid stain like EtBr. As a method to identify perturbations in the dsDNA helix, bisulfite modification can be used to detect R-loops [Classically, a series of electrophoresis techniques have been developed to identify sequences that favorably form R-loops cription , 23. Mor R-loops . Brieflyin vitro enhancement followed by sequencing (DRIVE-seq) utilizes an epitope-tagged, catalytically dead RNase H1 and affinity pulldown to recover DNA-RNA hybrids for sequencing [A powerful, genome-wide technique, known as DNA-RNA immunoprecipitation followed by sequencing (DRIP-seq), uses the S9.6 antibody to specifically pull down DNA-RNA hybrids that are subsequently identified by high throughput sequencing methods to map these structures to the genome -27. Whilquencing , 30. Depin vitro transcription. For example, the S9.6 antibody, as well as a GFP-tagged DNA-RNA hybrid binding domain of RNase H1, allow for visualization of R-loop foci with fluorescence microscopy [in situ hybridization (FISH) based techniques [Recently, IP-mass spectrometry techniques were used to identify R-loop interacting proteins in a high throughput format. The study identified the RNA helicase DHX9 and characterized its role in suppressing R-loop accumulation, reducing DNA damage, and promoting transcriptional termination . R-loopscroscopy , 32 or fchniques -35. Elecchniques .The continued development of techniques and methodologies to study R-loop dynamics and the downstream consequences of their accumulation will further detail the contribution that these structures have in gene regulation and genomic instability.in vivo role of R-loops as regulatory elements of specialized events in the eukaryotic cell. Previously, mitochondrial origins of replication contain R-loops that are used to facilitate replication of the mitochondrial DNA (mtDNA), a process likely conserved from prokaryotic ancestors [Predictably, formation of a DNA-RNA hybrid creates structural lesions in the genome that can affect replication, transcription, and recombination. Ongoing research continues to detail the ncestors . Studiesncestors , 38. In ncestors . Recentlncestors .While R-loops contribute to specialized events or structures in the human genome, they also have distinct roles in regulating transcription. In one instance, R-loops regulate epigenetic mechanisms of gene repression. When R-loops form in downstream CpG island promoter regions, they inhibit the activity of DNA methyltransferases to promote expression . In a seR-loop formation in G-Rich sequences downstream of the 3' polyadenylation (polyA) signal can promote RNA Pol II pausing . The R-lAccumulation of R-loops often results in increased cellular stress that leads to genomic instability. The current hypothesis states that stabilized, co-transcriptional R-loops disrupt RF progression during S-phase. This disruption causes stalling that can lead to fork collapse, DSBs, or incomplete replication before entry into mitosis. Recently, perturbation of the mRNA splicing, processing, and export factors have been shown to increase R-loop mediated genome instability. For example, disruption of the THO and Transcription Export (TREX) complex (discussed above) leads to a hyper-recombination phenotype in yeast and human cells , 44. IntThe deposition of R-loops at promoter and terminator regions has been shown to regulate initiation, pausing, and termination of transcription. Perturbation of these regulatory events could lead to inappropriate changes in gene expression or genetic stress. Studies indicate that mutation of the THO complex impairs elongation, possibly through the formation of R-loop intermediates that prevent RNA Pol II processivity . MoreoveDNA damage sensing and repair proteins have recently emerged as inhibitors of R-loop accumulation in eukaryotic cells  of BRCA2 in resolving R-loops has been difficult due to its contributions in DNA repair, RF stability, and other mechanisms associated with mitosis. Its inherent involvement in these processes may alone prevent R-loop formation by preventing RF collapse and recruiting the ssDNA binding protein, Rad51, to DSB sites . SeparatBoth BRCA1 (FANCS) and BRCA2 (FANCD1) are part of the Fanconi Anemia (FA) mediated DSB repair pathway. Therefore investigators elucidated that this complex may regulate genome stability through R-loop resolution. In addition to the protective functions of BRCA1 and BRCA2, the disruption of critical FA complex members FANCD2, FANCA, and FANCM lead to genomic instability and DNA damage from R-loop mediated RF collapse , 54. TheThe induction of stress due to R-loop accumulation largely results from collisions that occur between RFs and the transcriptional machinery, particularly in a head-on orientation . Genomicin vitro and associate with disease progression [R-loops can arise from a variety of cellular stresses, and lead to complications including replication defects, transcriptional irregularities, and genomic instability. The biological consequences of disrupted R-loop homeostasis has been described in multiple neurodegenerative diseases that largely associate with ataxia (Reviewed in ). GCrichgression , 59.R-loop induced disruption of gene expression occurs through distinct mechanisms in trinucleotide repeat disorders. Expansion of the repeat region in the first intron of FXN leads to R-loop formation, increased histone H3K9 dimethyl (H3K9me2) chromatin deposition, and impeded Pol II mediated transcription . ChemicaFragile X syndrome harbors a mechanism where trinucleotide expansion in the 5' UTR of the FMR1 gene leads to DNA methylation mediated silencing of this locus , 61. HowFurthermore, mutations in the machinery that maintain R-loop homeostasis also associate with neurodegenerative disease. Dysfunctional TREX or RNaseH2 are linked to Aicardi-Goutières Syndrome (AGS) while SETX mutations contribute to ataxia with oculomotor apraxia (AOA2) , 65. MutAs expected, the increased genome instability, chromosomal rearrangements, and DNA damage that occur during aberrant accumulation of R-loops is linked to oncogenesis , 67, 68 The Tudor containing protein 3 (TDRD3) plays a critical role in maintaining proper R-loop homeostasis at the c-MYC locus . TDRD3 rThis translocation commonly occurs in Burkitt's lymphoma and multiple myeloma and causes oncogenic levels of c-MYC expression. Loss-of-function in mRNP biogenesis machinery also associates with known cancers. For example, sequestration of the THO/TREX complex through Kaposi's sarcoma herpesvirus expression of ORF57 leads to increased genomic instability and subsequent malignant progression in immunodeficiency-associated models .Saccharomyces cerevisiae, the formation of R-loops at the noncoding telomere repeat-containing RNA (TERRA) locus facilitates type II survival in telomerase mutants [RAD52) dependent recombination events that result in long heterogenous tracts of telomeric repeats. Extended tracts of recombination dependent repeats appear in mammalian cells utilizing the alternative lengthening of telomere (ALT) pathway during bypass of senescence. Therefore, accumulation of R-loops at TERRA may also play a role in promoting ALT activation in some types of cancer.In  mutants . The surin vivo, we may discover new disease associations of R-loops.Together, these models provide a wide variety of mechanisms where stress associates with increased R-loop formation and can lead to progressive neurodegenerative disease or cancer. The diversity of these mechanisms lends credence to the importance of proper R-loop homeostasis. Redundancy in the machinery that resolves R-loops suggests these structures are dynamic and widespread (studies indicate that R-loops occupy up to 5% of the genome ). As we Emerging evidence demonstrates the importance of proper R-loop homeostasis. These structures provide critical regulatory functions for gene expression and, in some cases, gene rearrangements. However, their formation and resolution must be tightly regulated to prevent topological events that disrupt replication through impaired RF progression. Such impairment provides an avenue for genome instability and contributes to the development of progressive diseases like cancer. Moreover, prolonged R-loop occupancy exposes ssDNA to genotoxic stress, which produces deleterious effects on gene expression.Future examination of R-loops in cancer models should determine whether the seemingly independent functions of BRCA1 and BRCA2 are coordinated to prevent RF collapse through FANC-complex-mediated mechanisms. Specifically, research should elucidate whether BRCA1 recruits SETX to unwind R-loops while BRCA2 acts to traverse the lesion in a manner to complete replication and allow DNA repair to occur at a later phase. Moreover, delineating the coordination between RNase H, helicases, and topoisomerases to prevent R-loop accumulation could provide insight into why specific cell types are susceptible to diseases associated with improper R-loop homeostasis.Finally, the link between the epigenetics and sites of R-loop formation has been established, but further examination of the machinery that recruits chromatin-modifying enzymes is warranted. Discovery of mechanisms that link R-loop homeostasis to chromatin modification would lend further clues into the development of specific cancers or neurodegenerative diseases. Specifically, the field would benefit from identification of R-loop “readers” that allow cells to detect these structures and modify chromatin to repress gene expression or promote transcriptional termination.R-loops provide an interesting new source of replicative and transcriptional stress that clearly have disease-causing consequences. As new methodologies and models are developed to study their regulation and homeostasis, researches will continue to integrate this under-studied structure into cell biology and pathogenesis.


Despite recent improvements in imaging and therapy, prostate cancer (PCa) still causes substantial morbidity and mortality. In surgical treatment, incomplete resection of PCa and understaging of possible undetected metastases may lead to disease recurrence and consequently poor patient outcome. To increase the chance of accurate staging and subsequently complete removal of all cancerous tissue, prostate specific membrane antigen (PSMA) targeting agents may provide the surgeon an aid for the intraoperative detection and resection of PCa lesions. Two modalities suitable for this purpose are radionuclide detection, which allows sensitive intraoperative localization of tumor lesions with a gamma probe, and fluorescence imaging, allowing tumor visualization and delineation. Next to fluorescence, use of photosensitizers may enable intraoperative targeted photodynamic therapy to eradicate remaining tumor lesions. Since radiodetection and optical imaging techniques each have their own strengths and weaknesses, a combination of both modalities could be of additional value. Here, we provide an overview of recent preclinical and clinical advances in PSMA-targeted radio- and fluorescence-guided surgery of PCa. Despite recent improvements in imaging and therapy, PCa remains the most frequently diagnosed cancer type in men and is estimated to be the second leading cause of cancer related deaths 68Ga/18F-PSMA PET or nano-MRI scans Surgical treatment of PCa faces two main challenges. First, complete resection of the prostate tumor, and thus managing negative surgical margins, remains difficult In order to detect PCa, prostate-specific membrane antigen (PSMA), a type II transmembrane glycoprotein, represents an excellent target for several reasons. Firstly, PSMA is over-expressed on >90% of all primary PCa lesions, as well as tumor positive lymph nodes and distant metastasis. In contrast, PSMA expression on healthy tissues is minimal Pre- and intraoperative identification of malignant tissue could be improved using multiple techniques including radioguidance, fluorescence imaging, ultrasound- and/or MRI-guidance. In this review we will focus on and discuss the potential of the currently available PSMA-targeting agents for radio-, fluorescence- and multimodal-guided intraoperative detection of PCa lesions. In addition, the feasibility of near infrared (NIR) photosensitizer-conjugated PSMA-tracers for intraoperative targeted photodynamic therapy (tPDT) is discussed.Over the past five years, the intraoperative PSMA-radioguided surgery (RGS) approach entered the operating theater for PCa patients undergoing prostatectomy with or without extended pelvic lymphadenectomy. Radioguided lymph node dissections were applied in an experimental setting in patients both within the typical field of an extended lymph node template  and patients with atypically localized lesions  3 preoperatively 68Ga/18F-PSMA-PET imaging. Among patients that undergo sLND a biochemical response is reported in 40-60% of the patients and a complete biochemical response is only observed in 20-30% of the patients. One of the reasons for these disappointing results could be the incomplete surgical resections of all involved tumor positive lymph nodes PSMA-based SPECT/CT and PET/CT can accurately detect primary PCa and small lymph node metastasis up to a size of 0.5-1 cm99mTc-nanocolloid was first introduced for sentinel lymph node dissections. After injection in the prostate, the 99mTc-nanocolloid distributed to lymph nodes along the internal and external iliac vessels and the obturator fossa et al. 99mTc-nanocolloid (radioactive and fluorescent) In PCa surgery, radioguided resection using et al. used the [111In]In-DOTAGA-(3-iodo-y)-f-k-Sub(KuE)  detection of both primary and distant PSMA-positive tumors was made possible used the 11InIn-DOet al. demonstrated that 111In-PSMA-I&T based RGS allowed the resection of subcentimeter lesions and led to the removal of additional PCa lesions. In 2 patients positive lesions, as confirmed by histopathological analysis, were found that did not show up during preoperative [68Ga]Ga-HBED-CC-PSMA (68Ga-PSMA-11) PET/CT imaging et al. also successfully applied the 111In-PSMA-I&T ligand for radioguided resection of PSMA-positive lesions in an exemplary PCa patient  , timing of the surgery is crucial. However, the low energy gamma rays produced by 99mTc could be advantageous for RGS, as gamma probes have the highest sensitivity for predominantly lower energy gamma photon emitting radionuclides 68Ga-PSMA-11 PET/CT imaging, a high uptake of 99mTc-PSMA-I&S was observed 12 hours after injection, as measured by preoperative SPECT/CT imaging. This high uptake resulted in successful intraoperative detection and resection of radiosignal-positive lesions et al. compared the radioactive rating (positive vs. negative) of tissues resected during 99mTc-PSMA-I&S-RGS to their postoperative histopathological analysis 99mTc-PSMA-I&S-RGS all tumor lesions visualized on 68Ga-PSMA-11 PET could be resected and additional small metastasis, down to 3 mm in size, could be removed Maurer 111In-PSMA-I&T or 99mTc-PSMA-I&S mediated PSMA-RGS. In these studies PSA levels and time to biochemical recurrence were monitored 68Ga/18F-PSMA-PET imaging. However, designing these studies is challenging as surgical fields are difficult to define, outcomes of surgeries are hard to compare due to inter-surgeon quality differences, and the long survival of the patients hampers fast oncological outcome measurements. Nonetheless, larger patient numbers and long-term follow-up studies are needed to determine the clinical benefit of PSMA-RGS. These studies should include comparison of treatment-related complications, progression-free survival, overall survival and quality of life of PCa patients.Subsequent to the above mentioned studies multiple clinical follow up studies were performed using either ex vivo gamma-probe measurements. Ultimately, this might lead to improved oncological outcomes. Still, use of radioguidance could only solve one of the two major challenges in PCa surgery. Even though it might improve detection and removal of tumor positive lymph nodes, it is not an optimal aid for the surgeon in visualization and precise delineation of the primary tumor and hard-to-resect metastasis.Taken together, PSMA-RGS still represents an individual and not guideline-conform treatment concept without proven benefit on quality of life or cancer-specific survival, despite the large quantities of proof of principle data In recent years, an alternative intraoperative imaging method, near infrared fluorescence (NIRF) imaging, has entered the surgical theatre. Fluorescence imaging might be used to tackle the first challenge in the surgical treatment of PCa: achieving negative surgical resection margins in vivo targeting characteristics. A series of PSMA-targeted contrast agents have been developed by conjugating different NIR fluorophores to PSMA-targeting whole antibodies, antibody fragments and small molecules et al. who conjugated the highly potent PSMA inhibitor GPI [1] to IRDye78 [6] to create the molecule GPI-78  111In]In-DTPA-D2B-IRDye800CW conjugate, intraperitoneal LS174T PSMA-expressing tumors could be visualized specifically with both imaging modalities. Subsequent image-guided resection showed the feasibility of complete multimodal-guided resection of all intraperitoneal tumor lesions in vivo. Interestingly, in a mouse with several LS174T-PSMA tumors located at different depths in the peritoneal cavity, the limited penetration depth of NIRF imaging was demonstrated. Only the two most superficial tumors could be visualized with NIRF imaging, whereas microSPECT/CT imaging showed several additional tumor lesions Encouraging results were reported in several preclinical studies that have focused on the development of dual-labeled radionuclide and fluorescent PSMA-targeting agents. Lütje et al. developed a dual-labeled, glutamate-urea based, PSMA-targeting SPECT/NIRF imaging small molecule tracer conjugated with IRDye800CW and a DOTA chelator, labeled with 111In et al. developed a series of PSMA-11 targeting ligands conjugated with different fluorophores, among which the NIR IRDye800CW and Dylight800 68Ga-PSMA-11 agent (4.8 ± 1.3 %ID/g). Furthermore, proof of concept fluorescence-guided surgery studies with 68Ga-IRDye800CW-PSMA-11were performed in healthy pigs using a DaVinci robotic surgery system. A PSMA-specific fluorescent signal (1 hour p.i. of the tracer) was observed in the prostate, which expresses PSMA on a physiological level. This demonstrated the potential of the 68Ga-IRDye800CW-PSMA-11 ligands for fluorescence-guided radical prostatectomy . Based on the previous PSMA-I&T, with addition of the fluorophore Sulfo‐Cy5. PSMA-specific tracer uptake into LNCaP xenografts (4.5 ± 1.8 %ID/g) led to sufficient imaging contrast in 68Ga-PSMA-I&F PET and in intraoperative fluorescence imaging Very recently, Schottelius et al. showed that tumor targeted multimodal imaging using [111In]In-DOTA-girentuximab-IRDye800CW is safe and can be used for intraoperative guidance in patients with clear cell renal cell carcinoma So far, these preclinical studies demonstrate the potential value of multimodal strategies for image guidance during surgery. However, translation of the PSMA-targeting multimodal preclinical tracers to the clinical setting still has to be made. For other malignancies, multimodal tracers were translated to the clinic, indicating the potential of multimodal treatments in patients. For example, Hekman [1] and LC-Pyro [2]  to the murine anti-PSMA antibody D2B and subsequently labeled it with 111In. In 5 mice, intraoperative NIRF imaging could be used for guided resection of subcutaneous LS174T-PSMA tumor xenografts, demonstrating the applicability of the multimodal conjugate for intraoperative image-guided resection of tumor lesions. tPDT treatment of LS174T-PSMA tumor bearing mice using this tracer caused significant tumor growth delay, as tumors in the control group reached a size of 500 mm3 in 27.7 days (range: 19-42 days), while treated tumors reached this size after 50.4 days (range: 23-80 days). In addition, median survival of the treated mice (47 days) was significantly longer than that of the untreated mice (27days). This study provided the first proof-of-principle that multimodal [111In]In-DTPA-D2B-IRDye700DX can be used for pre- and intraoperative detection of PSMA positive tumors with radionuclide- and fluorescence-imaging, surgical guidance, and PSMA-targeted PDT. PSMA-targeted PDT for PCa therapy is still in its infancy, no clinical proof of concept studies have been performed yet. However, despite the fact that a lot of translational steps towards the clinic still need to be taken, PSMA-targeted PDT has a high potential to become a valuable therapeutic option especially to remove remaining tumor cells in PCa patients during and after surgery.In some cases, achieving complete resection of tumor tissue is challenging. For example when lymph nodes or positive tumor margins are located in close proximity to surrounding healthy tissue  ] Figure A feasibio Figure B-C 61-64] Figure D 65, 66.  High uptake in PSMA-positive tumor tissue, 2) good tumor retention up to the moment of surgery and 3) low uptake in background tissue. Sufficient tumor-to-background ratios are key for targeting and imaging of prostate cancer. Given the variety of available and clinically used PSMA tracers, it is difficult to reach consensus on which ligand, radionuclide and/or fluorophore combination is best suited for a particular application. Still, PSMA-targeting ligands should be designed to meet a specific clinical need. In case of intraoperative PCa detection several factors are important as will be discussed below.A lot of effort has been put into finding the optimal PSMA-binding small molecule, antibody fragment or whole antibody. Important properties of these PSMA-binding tracers are specificity for PSMA and sufficient accumulation and retention in the tumor to allow intraoperative detection. The first described PSMA binding tracers are monoclonal antibodies (mAbs) To overcome the disadvantages of mAbs and their derivates, small molecule PSMA-binding motifs with improved pharmacokinetics were developed 99mTc and 111In, which have several main advantages. First, besides intraoperative detection, these γ-emitters can be used for pre- and post-operative SPECT imaging. Second, 99mTc and 111In have half-lives of 6 hours and 2.8 days respectively, leading to sufficient time for preoperative SPECT scanning, multiple-hour and/or next day surgeries. The 6-hour half-life of 99mTc is better compatible with the fast pharmacokinetics of small molecule PSMA-tracers and could therefore be preferred over 111In. Last, the availability of the radionuclides is important. 99mTc is readily available in many hospitals, leading to a high availability even in regional hospitals 99mTc often is preferred over 111In because of its lower production costs, and shorter half-life, leading to lower radiation exposure to both patients and nuclear medicine personnel. Moreover, 99mTc emits lower energy gamma rays of 140 keV, that are preferred for collimation, and a greater activity can be administered per patient resulting in higher photon yields and better quality SPECT images In addition to choosing the optimal tracer for PSMA targeted intraoperative detection of PCa, it is of utmost importance to choose the most suitable imaging moieties. The most commonly used radionuclides for intraoperative gamma probe based PCa detection are 99mTc and 111In), RGS could also be performed using positron emitters such as 68Ga or 18F et al. reported poor performance of [18F]-FDG gamma probe-guided surgery in evaluating axillary lymph nodes in breast cancer patients, because of high background gamma levels from local and distant parts of the body Beside low-energy gamma emitting radionuclides  region of the spectrum (650-900 nm) are employed for intraoperative imaging. Main advantages of using these NIR dyes are a superior tissue penetration depth, reduced interference from the presence of blood in the surgical field and low auto-fluorescence of the tissue in the NIR region With the rapid growth of minimally-invasive laparoscopic and/or robot-assisted PCa surgery, there is a need for image-guidance technologies suitable for these procedures. One important development in fluorescence-guided robotic surgery is the introduction of the Intuitive Surgical's Firefly™ imaging technology  integrated in the da Vinci® robot.in vivo and ex vivo measurement of the radiosignal in minimally-invasive surgical procedures. However, these probes have limited maneuverability in vivo and therefore hamper the detection of low activity lesions that are located near high background areas such as the kidneys et al. have developed a DROP-IN gamma probe with an effective scanning direction range between 0-180°, that was proven to be a valuable tool for robot-assisted RGS procedures In RGS, laparoscopic gamma probes are used for et al. studied PSMA-expression in the neovasculature of 15 non-prostate tumors using five different PSMA mAbs. In this study a strong neovascular PSMA immunoreactivity was described in many tumors, including clear cell renal cell carcinoma, colonic adenocarcinoma, glioblastoma multiforme, non-small cell lung carcinoma and breast carcinoma  et al. the feasibility of pre- and postoperative imaging of gliomas using 68Ga-PSMA-11 PET/CT was investigated 68Ga-PSMA-11 scan and correlated with conventional MRI imaging. Furthermore, in a recent study by Meyer et al. the added value of pre-operative PSMA-based 18F-DCFPyL PET/CT imaging was suggested in patients with oligometastatic renal cell carcinoma 18F-DCFPyL PET/CT compared to conventional imaging, leading to detection rates of 88.9% and 66.7%, respectively.PSMA is expressed on the neovascular endothelium of numerous non-prostate solid tumors However, in other cancer types absolute tumor uptake of the PSMA-tracers is lower compared to PCa. Expression of PSMA is only present on the neovasculature, with little to no expression on the tumor cells and the normal vascular endothelium, possibly leading to lower detection rates. Therefore, PSMA-based imaging is most suitable for tumors with a high rate of neovascularization.The detection and removal of positive resection margins, small tumor lesions and micro-metastasis still poses major challenges in the surgical management of patients with PCa. Unfortunately, at the moment surgeons have to choose between complete oncological resection, which often leads to debilitating side effects, and nerve-saving operations focused on functional outcomes, with a higher chance of positive resection margins as a consequence 99mTc and 111In-labeled small molecule PSMA ligands, already used in a clinical studies, seem to be the most promising tracers for radioguided surgery. Currently, for PCa surgical removal of cancerous tissue is performed without any fluorescence image guidance. In the up-and-coming fluorescence-guided surgery field, NIR dye labeled PSMA ligands show encouraging results. In addition, photosensitizers like IRDye700DX could be used for tPDT of residual tumor lesions. Hence, use of such a theranostic dual-labeled PSMA ligand allows for imaging, resection and local killing of PCa cells.It is debatable which type of tracer is best suited for intra operative use, as both mAbs and small molecule PSMA tracers each have their own advantages, including high tumor uptake of mAbs and high specificity and fast pharmacokinetics of small molecule PSMA tracers. Modifications in small molecule PSMA-tracers, including linker length and addition of multiple negative charges, were found to alter the binding affinity of the PSMA ligands causing improved targeting characteristics and reduced background signals. At present, Presumably the most promising developments in the intraoperative field are the dual-labeling strategies that allow for both acoustic and visual detection of PSMA-expressing tumor lesions, micro-metastases and positive resection margins. Together, these multimodal strategies empower guided total resection of PCa tumors. Unfortunately, so far, fluorescent and dual-labeled PSMA-targeting tracers have only been investigated in a preclinical setting. In order to move the field forward, it is essential that proper prospective clinical studies are carried out measuring the additional value of multimodal image guided surgery compared to conventional surgery techniques. Clinical trials comparing these two treatments should be performed and main outcomes of these studies should include comparison of toxicology, complications, progression free survival, overall survival and quality of life of the PCa patients.


Nature Communications the group of Dr. Dmitry Gabrilovich takes us one step closer to understanding why lipid accumulation impairs the function of tumour-associated dendritic cells (DCs). In this study, the authors present two surprising and significant findings. First, they show that in mouse DCs oxidized lipids function as a sink that traps the heat shock chaperone HSP70, a molecular target of emerging anti-cancer strategies. Secondly, they find that HSP70 in turn regulates the trafficking of peptide-loaded major histocompatibility complex class I (pMHC-I) molecules, a complex that triggers the proliferation of cancer-killing T cells. These observations are discussed briefly in the context of lipid droplet function and pMHC-I trafficking in tumour-associated DCs, as well as HSP70’s pleiotropic and incompletely understood roles - and what they mean for future cancer therapy designs.In a recent publication in  Patients whose tumours harbour a greater number of DCs have a better prognosis [Dendritic cells (DCs) are antigen-presenting immune cells pivotal to anti-cancer immunity because of their unique ability to capture tumour-associated antigens and use them to activate cancer-killing cytolytic T cells. This process, termed cross-presentation, involves loading tumour-derived peptides onto major histocompatibility complex I (MHC-I) molecules. These peptide-MHC-I complexes (pMHC-I) then traffic to the cell surface, where together with co-stimulatory molecules they trigger the proliferation and differentiation CD8rognosis , yet clerognosis . Exactlyrognosis .increase DC lipid droplets [Notably in 2010 the Gabrilovich group observed an abnormal accumulation of lipid droplets  within tumour-associated DCs, which when inhibited, restored cross-presentation . Indeed droplets . Howeverdroplets . This obNature Communications, Veglia et al. now provide an unexpected potential mechanism involving the heat-shock-induced chaperone heat-shock protein 70 (HSP70) that might explain how oxidized lipid accumulation can lead to defective cross-presentation [oxidized lipid droplets essentially serve as a sink that traps cytosolic HSP70, preventing it from performing other functions.In their most recent paper published in entation . By combVeglia et al.This of course begs the next question, which is then how can HSP70 influence cross-presentation? Gabrilovich’s team begins to answer this with an astonishing observation that either inhibiting or depleting HSP70 leads to a re-routing of pMHC-I complexes, which rather than reaching the plasma membrane, are sent to lysosomes instead. Yet the remaining question of how HSP70 can regulate pMHC-I trafficking is perhaps the most intriguing, considering trafficking control is hardly a well-established role for the ubiquitous chaperone. HSP70 is best known for its protective role during cellular stress where it binds hydrophobic patches of unfolded proteins, helping them to refold and preventing their aggregation . Thus, wThus, a primary outstanding issue is whether it is intracellular or extracellular HSP70 that controls pMHC-I trafficking. It is curious to note that effects of HSP70 modulation were restricted to pMHC-I complexes, as the same was not observed when total MHC-I molecules were probed. Moreover, oxidized lipids influenced only the surface levels of pMHC-I bearing exogenous but not endogenous peptides . Taken tOverall, the research of Veglia and colleagues raises caution in employing HSP70 inhibitors as anti-cancer agents, as their use may prevent the initiation of anti-tumour immunity by DCs. On the other hand, deciphering which specific HSP70 forms are responsible for regulating pMHC-I complexes on DCs could help define whether tailoring such strategies to selectively target different HSP70 species could bypass these negative effects. It should be noted that the study was performed using mouse DCs and confirmation that similar effects occur in human cells would be beneficial. Additionally, this work suggests that lipid oxidation in the tumour microenvironment is a key factor in suppressing the anti-tumour capabilities of DCs, and favours therapeutic strategies including the administration of antioxidants such as vitamin E . Thus, a


Hantaviridae) in shrews , the once-conventional view that rodents (order Rodentia) served as the primordial reservoir hosts now appears improbable. The newly identified soricid-borne hantaviruses generally demonstrate well-resolved lineages organized according to host taxa and geographic origin. However, beginning in 2007, we detected sequences that did not conform to the prototypic hantaviruses associated with their soricid host species and/or geographic locations. That is, Eurasian common shrews (Sorex araneus), captured in Hungary and Russia, were found to harbor hantaviruses belonging to two separate and highly divergent lineages. We have since accumulated additional examples of these highly distinctive hantavirus sequences in the Laxmann’s shrew (Sorex caecutiens), flat-skulled shrew (Sorex roboratus) and Eurasian least shrew (Sorex minutissimus), captured at the same time and in the same location in the Sakha Republic in Far Eastern Russia. Pair-wise alignment and phylogenetic analysis of partial and full-length S-, M- and/or L-segment sequences indicate that a distinct hantavirus species related to Altai virus (ALTV), first reported in a Eurasian common shrew from Western Siberia, was being maintained in these closely related syntopic soricine shrew species. These findings suggest that genetic variants of ALTV might have resulted from ancient host-switching events with subsequent diversification within the Soricini tribe in Eurasia. With the recent discovery of genetically distinct hantaviruses (family  Suncus murinus), captured in southern India in 1964 [Apodemus agrarius) in Korea by more than a decade [Sorex araneus) and Eurasian water shrew (Neomys fodiens) in European Russia and the former Yugoslavia [Thottapalayam virus (TPMV), a previously unclassified virus isolated from an Asian house shrew , captured coincidentally as part of a HTNV surveillance program along the Imjin River near the demilitarized zone in South Korea, a genetically distinct hantavirus sharing a common ancestry with TPMV, named Imjin virus (MJNV), was isolated in 2008 [®-preserved and ethanol-fixed archival tissues from more than 50 shrew species  captured in Europe, Asia, Africa and North America during the past three decades (1980–2013), we have discovered multiple novel hantaviruses, which are more genetically diverse than those harbored by rodents [Guided by these decades-old reports and spurred by the fortuitous availability of tissues from Ussuri white-toothed shrews  [Neomys anomalus) ([Sorex daphaenodon) [Sorex tundrensis) [Among the shrew species in which HFRS antigens were originally detected more than 30 years ago, confirmation by RT-PCR detection of hantavirus RNA was first achieved in the Eurasian common shrew . The hanminutus) ,22,26, Maenodon)  and tunddrensis) .Phylogenetic analysis of SWSV and other newly identified soricid-borne hantaviruses show well-resolved lineages organized largely by host taxa and geographic origin ,9. HowevSorex species in Far Eastern Russia during the ensuing years forms the basis of this report. Also, in including expanded sequences of ALTV and previously unreported ALTV-like hantavirus sequences from two Eurasian common shrews from Hungary and one Eurasian pygmy shrew from Poland, as well as the recently reported full-length genome of Lena River virus (LENV) strain Khekhtsir-Sc67 from a Laxmann’s shrew (Sorex caecutiens) in Far Eastern Russia [Hantaviridae [Finding many more examples of these highly distinctive hantavirus sequences from syntopic n Russia , we demoaviridae .Shrew specimens were collected, as part of the Beringian Coevolution Project , along t2, 1 U of AmpliTaq polymerase  and 0.25 μM of each oligonucleotide primer [Total RNA was extracted from lung tissues, using the PureLink Micro-to-Midi total RNA purification kit , then reverse transcribed, using the SuperScript III First-Strand Synthesis System (Invitrogen) with random hexamers and universal oligonucleotide primer , designed from the conserved 3′ end of the S and L segments of hantaviruses ,16,20,26e primer ,20,23,27Pair-wise alignment and comparison of partial and full-length S- and L- and partial M-segment sequences of newfound hantaviruses from soricine shrews with representative rodent-, shrew-, mole- and bat-borne hantaviruses were performed, using the ClustalW method (TranslatorX server and BioEdit 7.0.5) ,36,37. Ihttp://web.cbio.uct.ac.za/~darren/rdp.html).Nucleotide sequences of the coding regions, including the full-length L segment, were analyzed using multiple recombination-detection methods ,39, inclThe maximum likelihood and Bayesian methods, implemented in RAxML Blackbox webserver  and MrBab sequences, along with published sequences for shrews and moles for this gene region, were generated, using the maximum-likelihood and Bayesian methods described previously [The taxonomic identity of the hantavirus-infected shrews was verified in genomic DNA extracted from tissues using the QIAamp DNA Mini Kit (Qiagen) and their phylogenetic relationships were studied by analysis of the complete 1140-nucleotide cytochrome b gene, amplified by PCR using well-tested primers  . Host pheviously ,46. The w/v) homogenates of lung tissues from Sorex shrews, confirmed as infected with hantavirus by RT-PCR and sequencing, were inoculated onto subconfluent monolayers of Vero E6 cells , grown in 25-cm2 flasks and maintained with Dulbecco’s minimum essential medium containing 5% fetal bovine serum. Cells were subcultured at two- to four-week intervals, at which time aliquots of cells were examined for hantavirus RNA by RT-PCR. Blind passages of cells were conducted for more than 100 days.Using the previously described methods ,47, 1% ap > 0.05). Of the 20 Sorex shrew-borne hantaviruses from the Sakha Republic, 14 resembled ALTV, instead of the expected host-specific hantavirus (SWSV and KKMV)  .Sorex shrews captured at three localities in the Sakha Republic. As noted earlier, hantavirus RNA was found predominantly in male shrews, with evidence of infection in only three female shrews. Pair-wise alignment and comparison of the full-length and partial S-, M- and L-segment sequences from Laxmann’s shrews and flat-skulled shrews showed three genetically distinct hantaviruses. The first was Kenkeme virus (KKMV) strain MSB148794, previously reported from the flat-skulled shrew [ed shrew . Howevered shrew . In addied shrew .The full-length 1287-nucleotide S-genomic segment of SWSV and KKMV from the Laxmann’s shrew and flat-skulled shrew, respectively, encoded a 428-amino acid nucleocapsid (N) protein and lacked the additional open reading frame encoding a nonstructural NSs protein, as determined by sequence alignment with cricetid rodent-borne orthohantaviruses. Nearly full-length S-segment sequences of 1164 to 1209 nucleotides, obtained for ALTV-like hantaviruses from three Laxmann’s shrews and one flat-skulled shrew, showed 51.1–53.9% and 46.4–47.9% sequence similarity at the nucleotide and amino acid levels, respectively, with KKMV and SWSV . By contPartial M-segment sequences, amplified from three Laxmann’s shrews  and a flat-skulled shrew (Sr424/MSB148679), also showed low level nucleotide sequence similarity of 61.3–65.6% with KKMV. By contrast, they exhibited 90.0–91.1% nucleotide sequence similarity and LENV Khekhtsir-Sc67 (which served as a surrogate for prototype ALTV Telet-Sa302 because M segment sequences were unavailable).Sorex shrews in the Sakha Republic.The nearly full-length L-genomic segments of 6303 to 6441 nucleotides, amplified from four Laxmann’s shrews  and a flat-skulled shrew (Sr424/MSB148679), showed 64.7–65.4% and 61.9–63.0% sequence similarity at the nucleotide and amino acid levels, respectively, with KKMV and SWSV . At the RDP4 Beta 4.36 failed to disclose any consistent evidence of recombination in the S- and L-genomic segments. Although separate regions of potential recombination were found in a few instances, there was no consistency or concordance between the detection methods, calling into question the validity of the identified sequences or the biological significance of recombination versus general heterogeneity in sequence evolutionary rates.Sorex araneus from Hungary (Sa122/MSB95363 and Sa123/MSB95469) and Finland (Uurainen/63L and Lohja/EWS10L) and Sorex caecutiens , and Sorex roboratus (Sr418/MSB148839 and Sr424/MSB148679) from Russia clustered with prototype ALTV Telet-Sa302 from Western Siberia  and Amga River (green)  and one flat-skulled shrew (Sr422/MSB148794) segregated into separate clades with SWSV and KKMV, respectively, while the other full-length and partial S-, M- and/or L-genomic sequences from 10 Laxmann’s shrews , three flat-skulled shrews  and one Eurasian least shrew (Smi458/MSB148651) consistently formed a highly divergent lineage with ALTV Telet-Sa302, which was more closely related to loanviruses and mobatviruses . Specifi Siberia . Similar Siberia . The ove (green) .Sorex shrew was molecularly confirmed by amplification and sequencing of the full-length 1140-nucleotide cytochrome b gene. Phylogenetic trees revealed well-supported lineages according to species.The identity of each hantavirus-infected Despite using well-established protocols involving multiple blind passages in Vero E6 cells for 100 days or longer, repeated attempts to isolate these highly divergent lineages of hantaviruses from archival frozen shrew tissues were unsuccessful.In 2007, we first detected highly divergent hantavirus sequences in Eurasian common shrews captured in Russia and Hungary. A partial L-segment sequence, named ALTV, was deposited in GenBank, but we were unwilling to report this until an expanded database was assembled. In this report, we demonstrate through genetic and phylogenetic analyses of full-length and near full-length sequences of the L- and S-genomic segments, as well as partial M-segment sequences, that genetic variants of ALTV are harbored and are being maintained in Laxmann’s shrews, flat-skulled shrews and Eurasian least shrews in the Sakha Republic in Far Eastern Russia, as well as in Eurasian common shrews in Western Siberia and Hungary. Importantly, these multiple strains of ALTV-like hantavirus are circulating simultaneously with the prototypic host-specific soricine shrew-borne orthohantaviruses. That is, Eurasian common shrews and Laxmann’s shrews harbor SWSV and flat-skulled shrews host KKMV, as well as ALTV-related hantaviruses in the same locality and at the same time.Apart from the Eurasian common shrew, the Laxmann’s shrew and the flat-skulled shrew, ALTV-related hantavirus sequences were also found in a Eurasian pygmy shrew, captured in Chmiel, in southeastern Poland. The Eurasian pygmy shrew, which is the principal reservoir host of Asikkala virus in Finland  and the Although the gender-specific prevalence of hantavirus infection was not statistically significant among soricine shrews in the Sakha Republic, there was a tendency toward a male predominance. This observation is consistent with the reported overrepresentation of hantavirus infection in male shrews and rodents, including the Ussuri white-toothed shrew , Norway Our overall findings confirm those of Ling and colleagues, who previously reported the co-existence of two genetically distinct hantavirus species—SWSV and ALTV-like hantavirus—circulating simultaneously in a single host species, the Eurasian common shrew, in Finland . Their cThe changing global landscape of hantaviruses has prompted a re-examination of previously long-held dogma about their host range, evolutionary origins and phylogeography ,9. BasedThe isolation of hantaviruses is fraught with difficulty, even from freshly collected tissues. Thus, while disappointing, it is not altogether surprising that we failed to isolate ALTV-like hantaviruses from frozen archival tissues stored since 2006. To date, only three hantaviruses have been isolated from non-rodent hosts: TPMV from the Asian house shrew ; MJNV frSorex araneus, Sorex caecutiens, Sorex minutissimus, Sorex minutus and Sorex roboratus. Co-circulation of hantaviruses in the same host species also raises the distinct possibility of co-infection and reassortment as a mechanism for rapid evolutionary change [Mammantavirinae. Whether this means there might be a fifth genus warrants further intensive investigation.Eurasian common shrews, Laxmann’s shrews and flat-skulled shrews, captured at the same time and in the same location in Hungary and Russia, were each found to harbor hantaviruses belonging to two separate and highly divergent lineages. Pair-wise alignment and phylogenetic analysis of partial and full-length S-, M- and/or L-segment sequences indicated the co-existence and maintenance of two distinct hantavirus lineages in these closely related syntopic soricine shrew species. These findings suggest possible ancient host-switching events from another reservoir. Although ALTV was originally detected in the Eurasian common shrew and ALTV-like hantaviruses have been found in Eurasian common shrews from Finland and Hungary, the primary reservoir host of ALTV is unknown. However, since the vast geographic distribution of ALTV-like hantaviruses coincides with the geographic range of the Laxmann’s shrew, this shrew species is the likely candidate. Alternatively, ALTV might represent an ancestral hantavirus lineage that may have subsequently diversified within the Soricini tribe in Eurasia, based on being detected in y change ,57,58,59


The high rates of bone marrow involvement by these lymphomas pose a major therapeutic challenge due to the chemotherapy-resistant nature of the bone marrow microenvironment and the limited utility of rituximab-based salvage regimens in patients with relapsed/refractory disease. Preclinical studies utilizing high-dose cyclophosphamide in combination with the anti-CD52 monoclonal antibody alemtuzumab have recently shown promise in the treatment of intramedullary disease, and a Phase I human trial is now underway. In support of such efforts, here we perform CD52 target validation on a series of double-hit (n = 40) and double-expressor (n = 58) lymphomas using immunohistochemistry. CD52 expression levels varied considerably across samples, however positive staining was observed in 75% of both double-hit and double-expressor lymphomas. Similarly, high levels of CD52 expression were seen in patients whose disease was associated with high-risk clinical features, including primary refractory status (73%), higher IPI score (76%), and bone marrow involvement (74%). CD52 expression was not significantly correlated with diagnostically relevant pathologic features such as morphology, cytogenetic findings or other immunophenotypic features, but was notably present in all cases lacking CD20 expression (n = 6). We propose that CD52 expression status be evaluated on a case-by-case basis to guide eligibility for clinical trial enrollment. MYC and BCL2 and/or BCL6 rearrangements  and the subset of diffuse large B-cell lymphomas (DLBCL) and HGBCL, not otherwise specified (NOS), with MYC and BCL2 protein over-expression  represent distinct but overlapping subsets of mature B-cell non-Hodgkin lymphomas with aggressive clinical course, poor response to conventional chemotherapy (i.e. R-CHOP) and high relapse rates . Among cases of DLBCL, NOS with double-expressor phenotype, the Hans cell-of-origin classification (GCB vs. non-GCB) showed no apparent effect on CD52 expression levels.Notably, patients previously treated with rituximab-based chemotherapy regimens frequently had CD52-positive disease (78%), including all 6 cases that were negative for CD20 expression. The results of additional immunohistochemical studies  showed no impact on CD52 expression status, and tumor morphology (blastoid vs. DLBCL vs. DLBCL/BL) was similarly non-predictive. Within the DHL group, the presence or absence of Treatment regimens incorporating the anti-CD20 monoclonal antibody rituximab have been the cornerstone of therapy for aggressive B-cell lymphomas for nearly two decades . DespiteThe presence and extent of extranodal involvement remain independent prognostic factors for patients with aggressive B-cell lymphomas . CertainMYC and BCL2 in mice reconstituted with human hematopoietic stem cells [A remarkable example of such influence comes from the Hemann laboratory at the Koch Institute/MIT, who recently developed a treatment-refractory humanized mouse model of DHL through the B-cell-specific co-expression of em cells . This stem cells . The co-em cells . The molem cells . The potem cells .th edition) [As nearly all studies investigating CD52 expression by aggressive B-cell neoplasms were performed prior to the establishment of our current working definitions of DHL and DEL, such studies failed to differentiate these more aggressive lymphomas from conventional DLBCL. Considering the many biological differences between these neoplasms, we believed it necessary to revisit this topic more formally following the recent release of the 2017 WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (revised 4edition) . The resFlow cytometric assessment of CD52 is now available in many clinical laboratories, but may not represent a universal replacement for IHC due to technical challenges that can impact the workup of DHL and DEL specimens. Several common features of aggressive B-cell lymphomas, including increased cell size, cell fragility and frequent association with sclerosis and necrosis, have the potential to result in non-diagnostic flow cytometry studies . FurtherS1 Table(XLSX)Click here for additional data file.


MicroRNAs are short non-coding RNAs that play a crucial role in the regulation of gene expression during cellular processes. The host-encoded miRNAs are known to modulate the antiviral defense during viral infection. In the last decade, multiple DNA and RNA viruses have been shown to produce miRNAs known as viral miRNAs (v-miRNAs) so as to evade the host immune response. In this review, we highlight the origin and biogenesis of viral miRNAs during the viral lifecycle. We also explore the role of viral miRNAs in immune evasion and hence in maintaining chronic infection and disease. Finally, we offer insights into the underexplored role of viral miRNAs as potential targets for developing therapeutics for treating complex viral diseases. Until the twenty first century, it was assumed that more than 95% of the eukaryotic genome is “junk” DNA; however, the advent of next-generation sequencing and high throughput functional screening has highlighted the regulatory functions of the non-coding genome. Around 50–85% of the mammalian genome is transcribed, with at least some non-coding RNA transcript, which includes microRNA (miRNA), siRNA, piwi-interacting RNA (piRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA) –4. In reMicroRNAs are small RNA molecules 18–22 nt) that play a crucial role in the regulation of gene expression by binding to the 3′ untranslated region (3′UTR) of target messenger RNAs (mRNAs) –7. miRNA–22 nt thIn this review, we summarize the miRNA-like non-coding RNAs encoded by DNA and RNA viruses and their roles in the evasion of host immunity. Finally, we discuss the possible role of these v-miRNAs as potential targets for developing therapeutics for the treatment of viral diseases.Two different approaches have been used to identify v-miRNAs. (i) The use of computational tools to predict the secondary structure of the precursor of v-miRNAs (pre-v-miRNAs). This approach usually results in a large number of false positives; however, it can lead to the identification of less abundant miRNA , 15. ii. The firStudies have indicated that viruses utilize the cellular machinery to encode miRNA. Similarly to eukaryotic miRNAs, v-miRNAs are generated by the Drosha and Dicer machinery , 22. Thein vitro , gastric carcinoma (GC), Hodgkin's lymphoma (HL), and nasopharyngeal carcinoma (NPC) –30. In iin vitro , 39.Following the identification of EBV-encoded miRNA, several reports identified miRNA expressed by Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8), another member of the γ-herpesvirus family. KSHV is known to cause Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), Multicentric Castleman's disease (MCD), and KSHV inflammatory cytokine syndrome –42. A toLike γ-herpesvirus, α- and β-herpesvirus are also found to express v-miRNAs. The β-herpesvirus human cytomegalovirus (HCMV) is commonly found in the human population and has the largest genome, 230 kb of double-stranded DNA (dsDNA), among the herpesvirus family . HCMV caHCMV miRs, namely, miR-UL-112-1, US25-1, US25-2, US25-2-5p, US5-1, US33-5p, and ULD148D, have been shown to inhibit HCMV DNA viral replication by targeting multiple host and viral regulatory genes (discussed in the following sections) –63. miR-Herpes simplex virus (HSV) has two members, HSV-1 and HSV-2, which are known to cause oral or genital herpes lesions . v-miRNAAlmost all herpesviruses encode their own v-miRNAs except varicella-zoster virus (VZV). Many small-RNA sequencing studies have been performed for VZV, but VZV v-miRNAs have not yet been identified .Human papillomaviruses (HPV) preferentially infect the keratinocytes of mucous membranes or skin and cause numerous benign and malignant lesions at different anatomical locations. HPV infection is a common cause of cervical cancer –84. HPV The first report for the prediction of HPV-encoded miRNAs came in 2011. In that study, the authors predicted the HPV-encoded miRNAs in several HPV types. They predicted the pre-miRNAs using a computational algorithm and compared the conserved mature miRNAs with currently known miRNAs. Predicted HPV miRNAs related to miR-466,-467, and -669 were common and specific to the mucosal HPV types. Also, the authors observed that HPV-38 expresses a miRNA conserved to human let-7a . In anotSimilarly, another study developed miRNA discovery by using a forced genome expression (miDGE) tool for the identification of miRNAs. The study screened 73 different PV genomes using miDGE and observed that most of the PV genomes are unlikely to encode viral miRNAs. However, they could identify and validate five different miRNAs . These HPV miRNAs targeted transcripts corresponding to the early region of the HPV genome .Hepadna virus family. Infection with HBV can cause chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). HBV has infected 2 billion people worldwide (Hepatitis B virus (HBV) is the best-known member of the orldwide . In comporldwide .To date, only one HBV-encoded miRNA has been identified by deep sequencing of HBV-positive HCC tissue. The corresponding study identified and confirmed the expression of HBV-miR-3. HBV-miR-3 downregulated HBV protein and HBV replication by reducing the expression of HBcAg, a positive regulator of HBV transcription and pregenomic RNA (pgRNA), which inhibits HBV replication overall .The adenovirus genome encodes two non-coding RNAs, namely VA-RNA I and VA-RNA II . VA RNAsPolyomavirus are non-enveloped viruses that infect a wide range of species including humans, primates, rodents, birds, and cattle . Few repMost of our current understanding of v-miRNAs has been attributed to v-miRNAs that were produced by DNA viruses. The detection of v-miRNAs in RNA viruses has been controversial, with a few reports suggesting non-canonical miRNA-like small RNAs produced during RNA virus infections; however, these small RNAs lack the canonical stem-loop structure found in miRNAs, so their biogenesis and function are not well-understood , 107. ThThe influenza virus replicates inside the host nucleus and it therefore differs from most of the other RNA viruses. As the virus replicates inside the host nucleus, it can utilize the nuclear miRNA processing factor Drosha to express v-miRNAs . One of Ebola virus (EBOV) is a negative-sense RNA virus that causes severe infectious disease with a high mortality rate of 83–90%. To date, few studies have identified miRNA-like small RNA encoded by EBOV –117. The+ PBMCs did not detect any HIV-1 miRNAs , UL112/113, and UL120/121 by translational inhibition rather than transcript degradation . miR-UL1EBV-encoded BART miRNAs play a key role in maintaining latency and controlling the viral life cycle. BART miRNAs such as miR-BART16, miR-BART17-5p, and miR-BART1-5p target the 3′ UTR of the LMP1 gene and negatively regulate LMP1 protein expression. LMP1 is capable of inducing cell growth and proliferation, but overexpression of LMP1 can result in inhibition of growth and apoptosis. Therefore, the downregulation of LMP1 expression may favor EBV-associated cancer development by exerting tight control on LMP1 expression . The EBVFew reports have identified v-miRNAs involved in the maintenance of KSHV latency. The KSHV-encoded miRNA miRK9 targets the viral protein RTA, a major lytic switch protein. RTA plays an important role in controlling viral reactivation from latency. v-miRNA-mediated regulation of RTA fine-tunes viral reactivation in the KSHV life cycle . In addiin silico tools, the HIV-1 genome was putatively shown to encode five pre-miRNAs. Based on the mature miRNA sequence deduced from the pre-miRNAs, these miRNAs were computationally predicted to target a large set of host cellular genes to establish a favorable cellular milieu for viral replication  have been identified in influenza virus infections that play a role in switching the viral polymerase from transcription toward genome replication by interacting with the polymerase machinery. However, whether svRNAs target viral or host transcripts is unknown . Using ilication .Evidence has been accumulating for v-miRNAs modulating the host immune response to enable a favorable intracellular milieu . To dateThe same study identified Epstein-Barr virus (EBV) miRNAs targeting host cellular genes in EBV-positive B cell lines. EBV miRNAs were shown to regulate cellular transport processes by targeting key genes such as TOMM22 and IPO7 . EBV miRHCMV miRNAs have been shown to target host genes involved in the antiviral immune response. miR-UL112 blocks the natural killer (NK) cell-mediated recognition of virus-infected cells by inhibiting the expression of MICB, a stress-induced ligand essential for NK-cell activity. Suppression of MICB results in decreased binding to the NKG2D receptor, thereby leading to decreased killing of virally infected cells by NK cells . InteresCombating viral diseases and virus-associated cancers is an ongoing health-related challenge present globally. Recent studies have focused on identifying miRNAs as targets for treating several diseases, including viral diseases. Antagomirs designed to sequester host miR-122 involved in HCV infection entered the phase II of human clinical trial and show promising effects against the infection , 181. UsSimilarly, using EBV promoters such as EBER2 promoter was reported to effectively express miRNA sponge to silence specific genes in EBV-infected cells and might be useful in the targeting of EBV-positive NPC cells . In addiAlthough the biogenesis, mechanism and function of virally encoded miRNAs are not well-characterized, substantial progress has been made in the last few years. With the emergence of high-throughput sequencing technologies and computational analysis tools, the number of newly discovered v-miRNAs is increasing. Multiple lines of evidence have strengthened the “classical” hypothesis of v-miRNAs solely originating from DNA viruses; however, some non-canonical miRNA-like RNA fragments have been detected during RNA virus infections. While the major functions of v-miRNAs across divergent virus families have been broadly attributed to immune evasion, autoregulation of the viral life cycle and tumorigenesis, there is still a broad gap in annotating the exact molecular determinants underlying these functions. The holy grail of the functional importance of v-miRNAs warrants more investigation to provide therapeutically amenable leads for targeting infectious diseases in the future.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ArtOn the Brain is a mobile application that incorporates art history education, play and socialization for older adults. This pilot project examined its health and social impact among long term care residents. A convenience sample of 48 residents, 60+ years, with borderline intact to moderate cognitive impairment were recruited. Residents with mild or better cognitive status used the app individually on a tablet while the remaining participated in therapist-led group sessions. One-hour intervention occurred twice weekly. Primary outcomes, health and well-being, were measured with 5Q-5D-5L and Warwick-Edinburgh Mental Well-being tools at baseline and after 6 weeks. Secondary outcomes were mood, cognition, function and overall satisfaction with activity options and personal relationships. Pre-post paired t-test analyses of data from EQ-5D-5L and Warwick tools did not show a significant difference. There were no notable declines with cognitive performance, functional ability and depression post-intervention. Four activity options and three personal relationship items had statistically significant improvements post-intervention. Activity options addressed engagement in enjoyable activities on weekends and evenings, and opportunity to, spend time with other like-minded residents and explore new skills, interests. Personal relationships items included engagement in activities helpful to others and having people to do things with. ArtOn the Brain is a novel application suitable for long term care residents. While 6 week intervention did not show improvement in overall health and well-being, there were significant improvements with residents’ reported engagement in activity options and improvement of personal relationships. Longitudinal evaluations are needed to examine changes in health status and well-being.


A 50-year-old man presented with dizziness and hearing disturbance in the right ear. Magnetic resonance imaging (MRI) revealed a well-enhanced mass lesion in the right cerebellopontine (CP) angle that appeared to originate in the cerebellum. A surgical specimen obtained at the subtotal resection with craniotomy revealed a diffuse large B-cell lymphoma (DLBCL). During the three courses of chemotherapy with high-dose methotrexate (MTX) with leucovorin rescue, he developed a right abducens palsy, left oculomotor palsy, left facial palsy, right trigeminal sensory disturbance, and paraparesis. Although the brain MRI showed that the CP angle tumor had disappeared completely following chemotherapy, enhanced lesions along the cauda equina were detected on a lumbar spine MRI. FDG-PET (18 F-fluorodeoxyglucose positron emission tomography) revealed multiple high-uptake abnormalities in the cranial nerves and spinal nerves. Tumor cells were found in the cerebrospinal fluid specimen from a lumbar puncture. Craniospinal irradiation was performed, including all the abnormal FDG high-uptake areas, and was effective in relieving the patient’s symptoms. On FDG-PET, the high-uptake abnormalities in the peripheral nerves disappeared. However, five weeks after the irradiation, he developed right trigeminal sensory disturbance, hoarseness, dysphagia, and right arm pain. FDG-PET disclosed multiple high-uptake abnormalities in more peripheral portions of the cranial nerves and spinal nerves. Chemotherapy with rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine (Oncovin®), and prednisolone (R-CHOP) was then resorted to which mitigated his symptoms. On follow-up FDG-PET, the high-uptake abnormalities in the peripheral nerves disappeared again. Primary non-Hodgkin’s lymphoma (NHL) of the central nervous system (CNS) is uncommon and generally affects the brain . DiffuseA 50-year-old man suffered from dizziness and hearing disturbance in the right ear. Magnetic resonance imaging (MRI) demonstrated a well-enhanced mass lesion in the right cerebellopontine (CP) angle, which appeared to have originated in the cerebellum Figure . The surgical specimen taken at the time of the subtotal resection with craniotomy revealed a DLBCL Figures -3.During the three courses of chemotherapy with high-dose methotrexate (MTX) with leucovorin rescue, he developed a right abducens palsy, left oculomotor palsy, left facial palsy, right trigeminal sensory disturbance, and paraparesis. Although the CP angle tumor on the brain MRI showed complete regression following chemotherapy Figure , enhanceFluorodeoxyglucose positron emission tomography (FDG-PET) revealed multiple high-uptake abnormalities in the cranial nerves and spinal nerves Figures -6.Tumor cells were found in the cerebrospinal fluid specimen from the lumbar puncture. Craniospinal irradiation was performed, including all the abnormal FDG high-uptake areas Figure , and effChemotherapy consisting of R-CHOP , and prednisolone) was then administered, which began two weeks after the development of recurrent symptoms. We chose R-CHOP, other than ordinal salvage regimens for CNS lymphoma such as high-dose cytosine arabinoside, because his general condition was not good enough after esophagitis was relieved. His symptoms again improved in two weeks after the beginning of R-CHOP. On the FDG-PET scan, taken three months after the beginning of R-CHOP (after three courses of R-CHOP), the high-uptake abnormalities in the peripheral nerves again disappeared. At that time his neurological conditions were good only with dysesthesia in the lower extremities.The lymphomatous infiltration of a nerve root  is known as NL or neurolymphomatosis . It is aHaydaroglu Sahin et al.  and KatyGd-enhanced MRI and FDG-PET are the imaging modalities of choice for evaluation of patients with lymphoma and suspected neural involvement . NL is dA case of primary central nervous system lymphoma presenting with peripheral nerve involvement was described. Recognizing these conditions with an understanding of the rare clinical entity of neurolymphomatosis is necessary for early diagnosis of similar cases. Detailed neurological examination and imaging, including FDG-PET, would be essential to grasp the extent of the disease. Timely assessment with chemotherapy and irradiation, if possible, might improve the patient's neurological status.


Myxococcus xanthus DK1622 is a rich source of novel secondary metabolites, and it is often used as an expression host of exogenous biosynthetic gene clusters. However, the frequency of obtaining large genome-deletion variants by using traditional strategies is low, and progenies generated by homologous recombination contain irregular deletions. The present study aims to develop an efficient genome-engineering system for this bacterium based on the Cre/loxP system. We first verified the functionality of the native cre system that was integrated into the chromosome with an inducible promoter PcuoA. Then we assayed the deletion frequency of 8-bp-spacer-sequence mutants in loxP by Cre recombinase which was expressed by suicide vector pBJ113 or self-replicative vector pZJY41. It was found that higher guanine content in a spacer sequence had higher deletion frequency, and the self-replicative vector was more suitable for the Cre/loxP system, probably due to the leaky expression of inducible promoter PcuoA. We also inspected the effects of different antibiotics and the native or synthetic cre gene. Polymerase chain reaction (PCR) and sequencing of new genome joints confirmed that the Cre/loxP system was able to delete a 466 kb fragment in M. xanthus. This Cre/loxP-mediated recombination could serve as an alternative genetic manipulation method. Myxococcus, the species Myxococcus xanthus is one of the best-studied organisms among myxobacteria, since its lifecycle can be investigated under laboratory conditions. The potential of M. xanthus strain DK1622 . Finally, the plasmids were confirmed by double digestion and sequencing.For the construction of suicide plasmid pBJ113-14 to insert lox661 into the downstream region of DK xanthene biosynthetic gene cluster, we used Red/ET recombination method. Briefly, we amplified the two homologous arms by two primer pairs S14E-451F which contains the pBJ113 vector multi-cloning site sequence and S14E-1457R, S14E-2057F which contains lox661 and the overlapped sequence of S14E-1457R and S14E-3649R which contains the pBJ113 vector multi-cloning site sequence. After purification of these two PCR products and linearized pBJ113 vector, these three fragments were simultaneously transformed into arabinose-induced ori region sequence (about 1.0 kb). Simultaneously, the integration efficiency by pSWU30 is more than tenfold that of homologous arm-containing pBJ113. To avoid the occurrence of homologous recombination at the ori region when we needed to use these two plasmids to integrate the same genome of DK1622 step by step, we adopted a strategy as described: firstly, the pBJ113 vector was used by kanamycin selection, and then used the pSWU30 vector by tetracycline selection. Therefore, to construct the mutant strains DK-30-cmR-XX-CCK, we firstly integrate the pBJ113-PcuoA-cre into DK1622 to give the strain DK-PcuoA-cre. Then pSWU30-cmR-XX series plasmids were electro-transformed into strain DK-PcuoA-creto obtain the series strains DK-30-cmR-XX-CCK, respectively. This is the process of obtaining cmR deletion strains and verifying the deletion efficiency via different types of mutant lox pairs by cre gene integrated into the chromosome genome and expressed by copper inducible.Site-specific integrative plasmid pSWU30 and suicide plasmid pBJ113 contain the same To investigate the deletion efficiency of different lox pairs by Cre recombinase expressed on shuttle plasmid pZJY41 in DK1622, we first construct the series strains DK-30-cmR-XX containing different lox pairs. A series of plasmids pSWU30-cmR-XX was electro-transformed into strain DK1622 to obtain the series strains DK-30-cmR-XX, respectively.To obtain the strain containing double mutant lox sites at different regions of the chromosome of DK1622, we used the suicide plasmid pBJ113-12which contained two homologous arms and lox712 designed between the two homologous arms, integrated into the genome of DK1622 by kanamycin selection. After confirming the correct integration by PCR amplification, then the double crossover occurred on CTT+galactose agar plates; the correct recombination clones were obtained by PCR amplification and sequencing. After purification, we got the lox712-containing strain DK-12 at the desired position. By the same method, we electro-transformed the plasmid pBJ113-14 into the strain DK-12 to obtain the strain DK-12-14 which contains a double mutant-type lox pair in the genome of DK1622 and at about the 468 kb region was flanked by the two lox sites.


A nanohybrid was employed for fingerprint imaging that was capable of simultaneous identification of individuals and TNT visualization in a “traffic-light” manner. i.e., the PAA-functionalized green QDs and red QD-doped silica nanoparticles, could be explored for fingerprint imaging. Due to the formation of a Meisenheimer complex between TNT and PAA, the green-emitting QDs could be quenched by TNT, meanwhile the red-emitting QDs were inert. Therefore, the nanohybrid exhibited a traffic light-type fluorescence color change (green-yellow-red) to TNT concentration in the range of 40–400 μM. This method is promising for potential applications in security-screening needs in public areas such as airports and train stations.Fingerprints are a unique characteristic of an individual. Recently, it has been realized that fingerprints carry more information about individuals than just their identity, for example, they may identify potential addicts and terrorists carrying explosives. Therefore, the development of imaging moieties capable of both fingerprint staining and drug/explosive visualization is of significant importance for forensic chemistry. Here we developed a nanohybrid comprising green- and red-emitting QDs for simultaneous fingerprint imaging and TNT visualization in fingerprints. The red-emitting Cu-doped ZnCdS (Cu–ZnCdS) QDs were embedded into silica nanoparticles and the green-emitting ZnCdS QDs were anchored onto the surface of the silica nanoparticles and further functionalized with polyallylamine (PAA). Both components of the nanohybrid,  Fingerprints are unique and invariable to an individual and hence are important for individual credentials, access control, and other forensic investigations, and are the cornerstone of forensic evidence. When a fingertip contacts a surface, residues are usually transferred to the substrate, thus leaving a latent fingerprint (LFP). As LFPs are invisible to the naked eye, they require either physical or chemical enhancement in order to make them visible and suitable for identification.Currently, fingerprints are very useful when police or other security agencies are able to obtain a positive match with those prints present in databases. Recently, it has been realized that fingerprints carry more information about individuals than just their identity,Herein, we present a dual-emitting quantum dot (QD) nanohybrid for LFP imaging that is capable of simultaneous individual identification and TNT visualization. Green-emitting ZnCdS QDs and red-emitting Cu-doped ZnCdS (Cu–ZnCdS) QDs were employed in this work because of the superior optical properties of QDs.In this research, ZnCdS QDs and Cu-doped ZnCdS QDs were employed to provide the green and red fluorescence colors, respectively. As shown in The nanohybrid of red QDs@silica@green QDs for LFP imaging and TNT visualization was fabricated by first doping of red-emitting QDs into silica nanoparticles and then covalent bonding of green-emitting QDs to the surface of the resultant silica nanoparticles  and ESI†The excellent optical properties of QDs have been extensively explored for LFP imaging.After the successful identification of individuals with both green- and red-emitting QDs through LFP imaging, we investigated their potential in simultaneous visual detection of TNT in colloidal solution . Selective interaction of TNT with primary amines has long been recognized for the development of various TNT sensors.After being functionalized with PAA to provide a primary amine, the fluorescence intensity of the green PAA–ZnCdS QDs could be gradually quenched upon the addition of TNT 2, AR), zinc nitrate (Zn(NO3)2, AR), sodium sulfide , sodium hydroxide , silver nitrate  and copper acetate (Cu(Ac)2, AR) were purchased from Chengdu Kelong Chemical Reagent Company . 3-Mercaptopropionic acid , l-cysteine , (3-aminopropyl)-triethoxysilane , 3-mercaptopropyltrimethoxysilane (MPS), ammonia solution , tetraethyl orthosilicate , polyallylamine , 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were obtained from Aladdin . 2,4,6-Trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT), nitrobenzene (NB) and paranitrotoluene (NT) were kindly supplied by Dr Wen-Sheng Zou (Anhui University of Architecture). All chemicals were used directly without further purification. Caution: the highly explosive TNT should be used with extreme caution and handled only in small quantities.Cadmium nitrate  and an F-7000 spectro-fluorometer , respectively. TEM and EDX characterization was performed with a Tecnai G2 F20 S-TWIN transmission electron microscope at an accelerating voltage of 200 kV . Samples for TEM were prepared by dropping a dilute solution of NPs onto nickel grids (for the Cu-doped QDs) or copper grids (for the ZnCdS QDs) with subsequent evaporation of the solvent in air at room temperature.All sebaceous fingerprints were acquired from the same volunteer. The hands were thoroughly washed with soap before fingerprint deposition. The fingertip was first dipped into TNT solutions of different concentrations and then gently rubbed across the forehead before stamping the finger onto the substrate, such as aluminum foil mainly. The foil strips bearing the fingerprints were immersed in the aqueous solution of dual-emission doped QDs for 30 s and taken out. The fingerprint images under UV lamp irradiation (365 nm) and other fluorescence images were photographed with a Nikon D300S digital camera equipping a Nikon AF-S VR 105mm f/2.8G IF-ED Macro Lens without any filters.Supplementary informationClick here for additional data file.