Pediatric Erythroid Sarcoma Diagnostically Confirmed by Identification of a Recurrent NFIA::CBFA2T3 Fusion.

Erythroid sarcoma (ES) is exceedingly rare in the pediatric population with only a handful of reports of de novo cases, mostly occurring in the central nervous system (CNS) or orbit. It is clinically and pathologically challenging and can masquerade as a nonhematopoietic small round blue cell tumor. Clinical presentation of ES without bone marrow involvement makes diagnosis particularly difficult. We describe a 22-month-old female with ES who presented with a 2-cm mass involving the left parotid region and CNS. The presence of crush/fixation artifact from the initial biopsy made definitive classification of this highly proliferative and malignant neoplasm challenging despite an extensive immunohistochemical workup. Molecular studies including RNA-sequencing revealed a NFIA::CBFA2T3 fusion. This fusion has been identified in several cases of de novo acute erythroid leukemia (AEL) and gene expression analysis comparing this case to other AELs revealed a similar transcriptional profile. Given the diagnostically challenging nature of this tumor, clinical RNA-sequencing was essential for establishing a diagnosis.

Flip the switch: the role of FleQ in modulating the transition between the free-living and sessile mode of growth in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen causing chronic infections that are associated with the sessile/biofilm mode of growth rather than the free-living/planktonic mode of growth. The transcriptional regulator FleQ contributes to both modes of growth by functioning both as an activator and repressor and inversely regulating flagella genes associated with the planktonic mode of growth and genes contributing to the biofilm mode of growth. Here, we review findings that enhance our understanding of the molecular mechanism by which FleQ enables the transition between the two modes of growth. We also explore recent advances in the mechanism of action of FleQ to both activate and repress gene expression from a single promoter. Emphasis will be on the role of sigma factors, cyclic di-GMP, and the transcriptional regulator AmrZ in inversely regulating flagella and biofilm-associated genes and converting FleQ from a repressor to an activator.

5-AZA Upregulates SOCS3 and PTPN6/SHP1, Inhibiting STAT3 and Potentiating the Effects of AG490 against Primary Effusion Lymphoma Cells.

Epigenetic modifications, including aberrant DNA methylation occurring at the promoters of oncogenes and oncosuppressor genes and histone modifications, can contribute to carcinogenesis. Aberrant methylation mediated by histone methylatransferases, alongside histones, can affect methylation of proteins involved in the regulation of pro-survival pathways such as JAK/STAT and contribute to their activation. In this study, we used DNA or histone demethylating agents, 5-Azacytidine (5-AZA) or DS-3201 (valemetostat), respectively, to treat primary effusion lymphoma (PEL) cells, alone or in combination with AG490, a Signal transducer and activator of transcription 3 (STAT3) inhibitor. Cell viability was investigated by trypan blue assay and FACS analysis. The molecular changes induced by 5-AZA and/or AG490 treatments were investigated by Western blot analysis, while cytokine release by PEL cells treated by these drugs was evaluated by Luminex. Statistical analyses were performed with Graphpad Prism® software (version 9) and analyzed by Student's t test or a nonparametric one-way ANOVA test. The results obtained in this study suggest that 5-AZA upregulated molecules that inhibit STAT3 tyrosine phosphorylation, namely Suppressor of Cytokine Signaling 3 (SOCS3) and tyrosine-protein phosphatase non-receptor type (PTPN) 6/Src homology region 2 domain-containing phosphatase-1 (SHP-1), reducing STAT3 activation and downregulating several STAT3 pro-survival targets in PEL cells. As this lymphoma is highly dependent on the constitutive activation of STAT3, 5-AZA impaired PEL cell survival, and when used in combination with AG490 JAK2/STAT3 inhibitor, it potentiated its cytotoxic effect. Differently from 5-AZA, the inhibition of the EZH1/2 histone methyltransferase by DS-3201, reported to contribute to STAT3 activation in other cancers, slightly affected STAT3 phosphorylation or survival in PEL cells, either alone or in combination with AG490. This study suggests that 5-AZA, by upregulating the expression level of SOCS3 and PTPN6/SHP1, reduced STAT3 activation and improved the outcome of treatment targeting this transcription factor in PEL cells.

Molecular Features of HHV8 Monoclonal Microlymphoma Associated with Kaposi Sarcoma and Multicentric Castleman Disease in an HIV-Negative Patient.

Human herpesvirus 8 (HHV8)-associated diseases include Kaposi sarcoma (KS), multicentric Castleman disease (MCD), germinotropic lymphoproliferative disorder (GLPD), Kaposi sarcoma inflammatory cytokine syndrome (KICS), HHV8-positive diffuse large B-cell lymphoma (HHV8+ DLBCL), primary effusion lymphoma (PEL), and extra-cavitary PEL (ECPEL). We report the case of a human immunodeficiency virus (HIV)-negative male treated for cutaneous KS, who developed generalized lymphadenopathy, hepatosplenomegaly, pleural and abdominal effusions, renal insufficiency, and pancytopenia. The excised lymph node showed features of concomitant involvement by micro-KS and MCD, with aggregates of HHV8+, Epstein Barr virus (EBV)-negative, IgM+, and lambda+ plasmablasts reminiscent of microlymphoma. Molecular investigations revealed a somatically hypermutated (SHM) monoclonal rearrangement of the immunoglobulin heavy chain (IGH), accounting for 4% of the B-cell population of the lymph node. Mutational analyses identified a pathogenic variant of KMT2D and variants of unknown significance in KMT2D, FOXO1, ARID1A, and KMT2A. The patient died shortly after surgery. The histological features (HHV8+, EBV-, IgM+, Lambda+, MCD+), integrated with the molecular findings (monoclonal IGH, SHM+, KMT2D mutated), supported the diagnosis of a monoclonal HHV8+ microlymphoma, with features intermediate between an incipient HHV8+ DLBCL and an EBV-negative ECPEL highlighting the challenges in the accurate classification of HHV8-driven lymphoid proliferations.

Manganese Acts as an Environmental Inhibitor of Pseudomonas aeruginosa Biofilm Development by Inducing Dispersion and Modulating c-di-GMP and Exopolysaccharide Production via RbdA.

The opportunistic human pathogen Pseudomonas aeruginosa causes chronic infections that involve multicellular aggregates called biofilms. Biofilm formation is modulated by the host environment and the presence of cues and/or signals, likely affecting the pool of the bacterial second messenger cyclic diguanylate monophosphate (c-di-GMP). The manganese ion Mn2+ is a divalent metal cation that is essential for pathogenic bacterial survival and replication during the infection in a host organism. In this study, we investigated how Mn2+ alters P. aeruginosa biofilm formation via the regulation of c-di-GMP levels. Exposure to Mn2+ was found to temporally enhance attachment but impair subsequent biofilm development, apparent by reduced biofilm biomass accumulation and lack of microcolony formation due to the induction of dispersion. Moreover, exposure to Mn2+ coincided with reduced production of the exopolysaccharides Psl and Pel, decreased transcriptional abundance of pel and psl, and decreased levels of c-di-GMP. To determine whether the effect of Mn2+ was linked to the activation of phosphodiesterases (PDEs), we screened several PDE mutants for Mn2+-dependent phenotypes (attachment and polysaccharide production) as well as PDE activity. The screen revealed that the PDE RbdA is activated by Mn2+ and is responsible for Mn2+-dependent attachment, inhibition of Psl production, and dispersion. Taken together, our findings suggest Mn2+ is an environmental inhibitor of P. aeruginosa biofilm development that acts through the PDE RbdA to modulate c-di-GMP levels, thereby impeding polysaccharide production and biofilm formation but enhancing dispersion. IMPORTANCE While diverse environmental conditions such as the availability of metal ions have been shown to affect biofilm development, little is known about the mechanism. Here, we demonstrate that Mn2+ affects Pseudomonas aeruginosa biofilm development by stimulating phosphodiesterase RbdA activity to reduce the signaling molecule c-di-GMP levels, thereby hindering polysaccharide production and biofilm formation but enhancing dispersion. Our findings demonstrate that Mn2+ acts as an environmental inhibitor of P. aeruginosa biofilms, further suggesting manganese to be a promising new antibiofilm factor.

Evolutionary flexibility in routes to mat formation by Pseudomonas.

Many bacteria form mats at the air-liquid interface of static microcosms. These structures typically involve the secretion of exopolysaccharides, the production of which is often controlled by the secondary messenger c-di-GMP. Mechanisms of mat formation have been particularly well characterized in Pseudomonas fluorescens SBW25; stimuli or mutations that increase c-di-GMP production by diguanylate cyclases (WspR, AwsR, and MwsR) result in the secretion of cellulose and mat formation. Here, we characterize and compare mat formation in two close relatives of SBW25: Pseudomonas simiae PICF7 and P. fluorescens A506. We find that PICF7-the strain more closely related to SBW25-can form mats through mutations affecting the activity of the same three diguanylate cyclases as SBW25. However, instead of cellulose, these mutations activate production of the exopolysaccharide Pel. We also provide evidence for at least two further-as yet uncharacterized-routes to mat formation by PICF7. P. fluorescens A506, while retaining the same mutational routes to mat formation as SBW25 and PICF7, preferentially forms mats by a semi-heritable mechanism that culminates in Psl and Pga over-production. Our results demonstrate a high level of evolutionary flexibility in the molecular and structural routes to mat formation, even among close relatives.

Role of Pel and Psl polysaccharides in the response of Pseudomonas aeruginosa to environmental challenges: oxidative stress agents (UVA, H2O2, sodium hypochlorite) and its competitor Staphylococcus aureus.

Pseudomonas aeruginosa is a versatile bacterium capable of adapting to a wide range of stress factors, including solar UVA radiation (400-315 nm). High UVA doses produce lethal effects due to the action of reactive oxygen species. Sublethal UVA doses also induces oxidative damage, but, in addition, it triggers a variety of adaptive responses, including the overexpression of pelA and pslA genes in P. aeruginosa. These genes encode the synthesis of Pel and Psl, which are essential polysaccharides in biofilm formation. The present study analysed the role of Pel and Psl in the adaptive responses generated by exposure to low UVA doses, and their importance in the response to lethal doses of UVA, hydrogen peroxide (H2O2), and sodium hypochlorite, in both planktonic cells and submerged and air-liquid interface (ALI) biofilms. It also studied the roles of Pel and Psl in P. aeruginosa-Staphylococcus aureus interaction. The results demonstrate that the capacity of sublethal UVA exposure to increase cell hydrophobicity and cell attachment and generate cross-protection phenomena in P. aeruginosa depends on the presence of Pel and Psl. The study also shows that Pel and Psl have a key role in the tolerance to lethal doses of UVA radiation, sodium hypochlorite and H2O2, in both biofilms and planktonic cells. Finally, co-culture assays showed total inhibition of S. aureus growth in presence of P. aeruginosa. This phenomenon depends, at least in part, on the simultaneous presence of Pel and Psl in planktonic cells and biofilms, suggesting a relevant role of these polysaccharides in the interaction between these species.

KSHV RTA Induces Degradation of the Host Transcription Repressor ID2 To Promote the Viral Lytic Cycle.

The immediate early viral protein replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for activating the lytic cycle of KSHV. RTA induces the KSHV lytic cycle by several mechanisms, acting as a viral transcription factor that directly induces viral and host genes and acting as a viral E3 ubiquitin ligase by degrading host proteins that block viral lytic replication. Recently, we have characterized the global gene expression changes in primary effusion lymphoma (PEL) upon lytic reactivation of KSHV, which also led to the identification of rapidly downregulated genes such as ID2, an inhibitor of basic helix-loop-helix transcription factors. Here, we demonstrate that ID2 overexpression in PEL ablates KSHV lytic reactivation, indicating that ID2 inhibits the KSHV lytic cycle. Furthermore, we show that while ID2 is highly expressed during latency, its protein level is rapidly reduced by 4 h postinduction during lytic reactivation. Our results indicate that RTA binds to ID2 and induces its degradation during the KSHV lytic cycle by N-terminal ubiquitination through the ubiquitin-proteasome pathway. Importantly, we found that not only KSHV RTA but also its Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) homologs interact with ID2, and they can induce the degradation of all four members of the ID protein family, suggesting an evolutionarily conserved interplay between gammaherpesvirus RTAs and ID proteins. Taken together, we propose that ID2 acts as a repressor of the KSHV lytic cycle, which is counteracted by its RTA-mediated degradation. We also predict that ID proteins may act as restriction factors of the lytic phase of the other gammaherpesviruses as well. IMPORTANCE In addition to its transcription regulatory role, RTA is also known to have an E3 ubiquitin ligase activity, which RTA utilizes for inducing protein degradation. However, it is still largely unknown what host factors are downregulated during KSHV lytic reactivation by RTA-mediated protein degradation and what the biological significance of the degradation of these host factors is. In this study, we discovered that RTA employs N-terminal ubiquitination to induce degradation of ID2, a potent transcription repressor of host genes, via the ubiquitin-proteasome pathway to promote KSHV lytic reactivation in PEL cells. Furthermore, we found that not only KSHV RTA but also RTA of EBV and MHV68 gammaherpesviruses can induce the degradation of all four human ID proteins, indicating that the interplay between gammaherpesvirus RTAs and ID proteins is evolutionarily conserved.

SUMO Modification of Histone Demethylase KDM4A in Kaposi's Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma.

Primary effusion lymphoma (PEL) is a fatal B-cell lymphoma caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Inducing KSHV lytic replication that causes the death of host cells is an attractive treatment approach for PE; however, combination therapy inhibiting viral production is frequently needed to improve its outcomes. We have previously shown that the KSHV lytic protein K-bZIP can SUMOylate histone lysine demethylase 4A (KDM4A) at lysine 471 (K471) and this SUMOylation is required for virus production upon KSHV reactivation. Here, we demonstrate that SUMOylation of KDM4A orchestrates PEL cell survival, a major challenge for the success of PEL treatment; and cell movement and angiogenesis, the cell functions contributing to PEL cell extravasation and dissemination. Furthermore, integrated ChIP-seq and RNA-seq analyses identified interleukin-10 (IL-10), an immunosuppressive cytokine, as a novel downstream target of KDM4A. We demonstrate that PEL-induced angiogenesis is dependent on IL-10. More importantly, single-cell RNA sequencing (scRNA-seq) analysis demonstrated that, at the late stage of KSHV reactivation, KDM4A determines the fates of PEL cells, as evidenced by two distinct cell populations; one with less apoptotic signaling expresses high levels of viral genes and the other is exactly opposite, while KDM4A-K417R-expressing cells contain only the apoptotic population with less viral gene expression. Consistently, KDM4A knockout significantly reduced cell viability and virus production in KSHV-reactivated PEL cells. Since inhibiting PEL extravasation and eradicating KSHV-infected PEL cells without increasing viral load provide a strong rationale for treating PEL, this study indicates targeting KDM4A as a promising therapeutic option for treating PEL. IMPORTANCE PEL is an aggressive and untreatable B-cell lymphoma caused by KSHV infection. Therefore, new therapeutic approaches for PEL need to be investigated. Since simultaneous induction of KSHV reactivation and apoptosis can directly kill PEL cells, they have been applied in the treatment of this hematologic malignancy and have made progress. Epigenetic therapy with histone deacetylase (HDAC) inhibitors has been proved to treat PEL. However, the antitumor efficacies of HDAC inhibitors are modest and new approaches are needed. Following our previous report showing that the histone lysine demethylase KDM4A and its SUMOylation are required for lytic reactivation of KSHV in PEL cells, we further investigated its cellular function. Here, we found that SUMOylation of KDM4A is required for the survival, movement, and angiogenesis of lytic KSHV-infected PEL cells. Together with our previous finding showing the importance of KDM4A SUMOylation in viral production, KDM4A can be a potential therapeutic target for PEL.

Improving the specific activity and stability of alkaline pectinase PEL3 through SpyTag/SpyCatcher cyclization.

OBJECTIVES: PEL3, an alkaline pectinase, exhibited the highest activity among documented alkaline pectate lyases reported in our early study. Unfortunately, undesired thermal stability hampering its industrial application. The purpose of this study is to enhance the performance of wild-type PEL3 (W-PEL3) based on SpyTag/SpyCatcher-mediated cyclization. RESULTS: The cyclized PEL3 (C-PEL3) was observed to fold correctly and generate a spatial conformation in a head-to-tail manner in E. coli. C-PEL3 exhibited comparable optimum pH and temperature to those of W-PEL3. Moreover, the catalytic activity of C-PEL3 increased by 23% compared to W-PEL3, and the kcat/Km of C-PEL3 was 1.5-fold greater than that of the W-PEL3. Importantly, C-PEL3 showed improved stability compared to W-PEL3. Firstly, C-PEL3 displayed a 65% increase in residual activity after treatment at 55 °C for 30 min. Secondly, C-PEL3 was prone to resist heat-induced protein aggregation. Thirdly, C-PEL3 exhibited metal ion stability. Circular dichroism analysis revealed that C-PEL3 was more capable of maintaining its secondary structures than W-PEL3 upon heat treatment. CONCLUSIONS: C-PEL3, the initial example of a circular pectinase through SpyTag/SpyCatcher cyclization, exhibits superior performance and represents a highly encouraging contender for industrial utilization.

HSPs/STAT3 Interplay Sustains DDR and Promotes Cytokine Release by Primary Effusion Lymphoma Cells.

Primary effusion lymphoma (PEL) is a rare and aggressive B-cell lymphoma, against which current therapies usually fail. In the present study, we show that targeting HSPs, such as HSP27, HSP70 and HSP90, could be an efficient strategy to reduce PEL cell survival, as it induces strong DNA damage, which correlated with an impairment of DDR. Moreover, as HSP27, HSP70 and HSP90 cross talk with STAT3, their inhibition results in STAT3 de-phosphorylation and. On the other hand, the inhibition of STAT3 may downregulate these HSPs. These findings suggest that targeting HSPs has important implications in cancer therapy, as it can reduce the release of cytokines by PEL cells, which, besides affecting their own survival, could negatively influence anti-cancer immune response.

NFE2L2 and STAT3 Converge on Common Targets to Promote Survival of Primary Lymphoma Cells.

NFE2L2 and STAT3 are key pro-survival molecules, and thus, their targeting may represent a promising anti-cancer strategy. In this study, we found that a positive feedback loop occurred between them and provided evidence that their concomitant inhibition efficiently impaired the survival of PEL cells, a rare, aggressive B cell lymphoma associated with the gammaherpesvirus KSHV and often also EBV. At the molecular level, we found that NFE2L2 and STAT3 converged in the regulation of several pro-survival molecules and in the activation of processes essential for the adaption of lymphoma cells to stress. Among those, STAT3 and NFE2L2 promoted the activation of pathways such as MAPK3/1 and MTOR that positively regulate protein synthesis, sustained the antioxidant response, expression of molecules such as MYC, BIRC5, CCND1, and HSP, and allowed DDR execution. The findings of this study suggest that the concomitant inhibition of NFE2L2 and STAT3 may be considered a therapeutic option for the treatment of this lymphoma that poorly responds to chemotherapies.

Primary Effusion Lymphoma, Multicentric Castleman's Disease, and Kaposi's Sarcoma in an HHV-8 and HIV-Positive Patient: A Case Report.

Primary effusion lymphoma (PEL), Kaposi's sarcoma (KS), and multicentric Castleman's disease (MCD) is an uncommon group of diseases included in the same spectrum with related characteristics. The coexistence of all of them in the same individual is a rare occurrence. We present the case of a 25-year-old patient diagnosed with human immunodeficiency virus (HIV) and the development of all these related pathologies. Despite the use of intensive treatment according to the latest recommendations, the evolution was unfavorable. This case reflects the need for new therapies and research in this field.

Nonmotile Subpopulations of Pseudomonas aeruginosa Repress Flagellar Motility in Motile Cells through a Type IV Pilus- and Pel-Dependent Mechanism.

The downregulation of Pseudomonas aeruginosa flagellar motility is a key event in biofilm formation, host colonization, and the formation of microbial communities, but the external factors that repress motility are not well understood. Here, we report that on soft agar, swarming motility can be repressed by cells that are nonmotile due to the absence of a flagellum or flagellar rotation. Mutants that lack either flagellum biosynthesis or rotation, when present at as little as 5% of the total population, suppressed swarming of wild-type cells. Non-swarming cells required functional type IV pili and the ability to produce Pel exopolysaccharide to suppress swarming by the flagellated wild type. Flagellated cells required only type IV pili, but not Pel production, for their swarming to be repressed by non-flagellated cells. We hypothesize that interactions between motile and nonmotile cells may enhance the formation of sessile communities, including those involving multiple genotypes, phenotypically diverse cells, and perhaps other species. IMPORTANCE Our study shows that, under the conditions tested, a small population of non-swarming cells can impact the motility behavior of a larger population. The interactions that lead to the suppression of swarming motility require type IV pili and a secreted polysaccharide, two factors with known roles in biofilm formation. These data suggest that interactions between motile and nonmotile cells may enhance the transition to sessile growth in populations and promote interactions between cells with different genotypes.

Pseudomonas aeruginosa Oligoribonuclease Controls Tolerance to Polymyxin B by Regulating Pel Exopolysaccharide Production.

Polymyxins are considered as the last resort antibiotics to treat infections caused by multidrug-resistant Gram-negative pathogens. Pseudomonas aeruginosa is an opportunistic pathogen that causes various infections in humans. Proteins involved in lipopolysaccharide modification and maintaining inner and outer membrane integrities have been found to contribute to the bacterial resistance to polymyxins. Oligoribonuclease (Orn) is an exonuclease that regulates the homeostasis of intracellular (3'-5')-cyclic dimeric GMP (c-di-GMP), thereby regulating the production of extracellular polysaccharide in P. aeruginosa. Previously, we demonstrated that Orn affects the bacterial resistance to fluoroquinolone, ß-lactam and aminoglycoside antibiotics. In this study, we found that mutation of orn increased the bacterial survival following polymyxin B treatment in a wild-type P. aeruginosa strain PA14. Overexpression of c-di-GMP degradation enzymes in the orn mutant reduced the bacterial survival. By using a fluorescence labeled polymyxin B, we found that mutation of orn increased the bacterial surface bound polymyxin B. Deletion of the Pel synthesis genes or treatment with a Pel hydrolase reduced the surface bound polymyxin B and bacterial survival. We further demonstrated that Pel binds to extracellular DNA (eDNA), which traps polymyxin B and thus protects the bacterial cells. Collectively, our results revealed a novel defense mechanism against polymyxin in P. aeruginosa.

Preclinical Evaluation of Recombinant Microbial Glycoside Hydrolases as Antibiofilm Agents in Acute Pulmonary Pseudomonas aeruginosa Infection.

The bacterium Pseudomonas aeruginosa can colonize the airways of patients with chronic lung disease. Within the lung, P. aeruginosa forms biofilms that can enhance resistance to antibiotics and immune defenses. P. aeruginosa biofilm formation is dependent on the secretion of matrix exopolysaccharides, including Pel and Psl. In this study, recombinant glycoside hydrolases (GHs) that degrade Pel and Psl were evaluated alone and in combination with antibiotics in a mouse model of P. aeruginosa infection. Intratracheal GH administration was well tolerated by mice. Pharmacokinetic analysis revealed that, although GHs have short half-lives, administration of two GHs in combination resulted in increased GH persistence. Combining GH prophylaxis and treatment with the antibiotic ciprofloxacin resulted in greater reduction in pulmonary bacterial burden than that with either agent alone. This study lays the foundation for further exploration of GH therapy in bacterial infections.

The impairment of DDR reduces XBP1s, further increasing DNA damage, and triggers autophagy via PERK/eIF2alpha in MM and IRE1alpha/JNK1/2 in PEL cells.

Cancer cells, particularly MM, that are highly secretory cells, and PEL cells that harbor KSHV, are characterized by high level of stress to which they adapt by activating DDR, UPR and autophagy. It is known that UPR sensors may affect DDR, but whether DDR manipulation influences UPR is less known. In this study, we found an intricate interplay between these responses. Indeed, PARP and CHK1 inhibition by AZD2461 and UCN-01, by downregulating c-Myc, reduced the expression of XBP1s, constitutively expressed in these cells, and upregulated CHOP. Interestingly, given the role of XBP1s in regulating DDR, BRCA-1 expression level was reduced, exacerbating DNA damage. Finally, DDR/UPR interplay activated a pro-survival autophagy via PERK/eIF2alpha axis in MM and IRE1alpha/JNK axis in PEL cells, since in the latter case PERK/eIF2alpha activation could be prevented by KSHV that, as other herpesviruses, tries to avoid the blocks of protein translation that this pathway may induce.

The ORF45 Protein of Kaposi Sarcoma-Associated Herpesvirus Is an Inhibitor of p53 Signaling during Viral Reactivation.

Kaposi sarcoma-associated herpesvirus (KSHV) is a carcinogenic double-stranded DNA virus and the etiological agent of Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). To prevent premature apoptosis and support its replication cycle, KSHV expresses a series of open reading frames (ORFs) that regulate signaling by the p53 tumor suppressor protein. Here, we describe a novel viral inhibitor of p53 encoded by KSHV ORF45 and identify its mechanism of action. ORF45 binds to p53 and prevents its interactions with USP7, a p53 deubiquitinase. This results in decreased p53 accumulation, localization of p53 to the cytoplasm, and diminished transcriptional activity. IMPORTANCE Unlike in other cancers, the tumor suppressor protein p53 is rarely mutated in Kaposi sarcoma (KS). Rather, Kaposi sarcoma-associated herpesvirus (KSHV) inactivates p53 through multiple viral proteins. One possible therapeutic approach to KS is the activation of p53, which would result in apoptosis and tumor regression. In this regard, it is important to understand all the mechanisms used by KSHV to modulate p53 signaling. This work describes a novel inhibitor of p53 signaling and a potential drug target, ORF45, and identifies the mechanisms of its action.

Ibuprofen involves with the reduced expression of pelD and pelF in pathogenic Pseudomonas aeruginosa strains.

Biofilm formation is an important factor in disease development by Pseudomonas aeruginosa. Similar to many bacterial species, biofilm formation in P. aeruginosa is regulated by the bacterial quorum sensing system. The pel genes are responsible for the synthesis of a glucose-rich polysaccharide that is associated with biofilm initiation and maturation. The antibiofilm potential of ibuprofen has been reported; however, the effect of the drug on the expression of the genes involved with biofilm formation has rarely been described. In this work, the effect of ibuprofen on the biofilm formation and expression of pelD and pelF genes among pathogenic P. aeruginosa strains was investigated. Multiple drug-resistant P. aeruginosa strains were treated with ibuprofen at ½ MIC concentration and their biofilm formation and expression of pelD and pelF genes was determined using the crystal violet and real-time PCR assays, respectively. The results showed that the ibuprofen at 1024 µg/mL significantly reduced biofilm formation of P. aeruginosa strains by 52-77%, compared with the controls. In addition, treating the bacteria with ibuprofen decreased the expression of pelD and pelF genes to 0.56 and 0.69 folds, respectively. We hypothesized that the attenuation of the pel genes could be associated with the reduction of bacterial QS autoinducers, which in turn reduced cellular c-di-GMP level. This work suggests that ibuprofen is a potent antibiofilm drug that could be used to enhance bacterial susceptibility to antimicrobials through the inhibition of biofilm formation.

Kaposi sarcoma (KS) with primary effusion lymphoma in HIV infected MSM (men having sex with men) co-infected with pulmonary tuberculosis and syphilis: a case report from India.

We describe a case of a 30-year-old MSM recently diagnosed with HIV, immunocompromised with a purplish or brown rash all over the body for 3 to 4 months. The histopathology of the cutaneous lesions and pleural effusion aspirate confirmed the diagnosis of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). While KS is one of the AIDS-defining illnesses seen in immunocompromised patients having low CD4 count, PEL is a rare and distinct subset of AIDS-related lymphoma. Despite the widespread availability of HIV testing, HIV diagnosis gets delayed due to stigma among MSM. This case report emphasizes the importance of early suspicion for symptoms of HIV-associated opportunistic infections in high-risk populations like MSM. The report reiterates the need for an ambient stigma-free environment for improving HIV screening in this high-risk population.