CRISPR knockout genome-wide screens identify the HELQ-RAD52 axis in regulating the repair of cisplatin-induced single stranded DNA gaps.

Treatment with genotoxic agents, such as platinum compounds, is still the mainstay therapeutical approach for the majority of cancers. Our understanding of the mechanisms of action of these drugs is however imperfect, and continuously evolving. Recent advances in the field highlighted single stranded DNA (ssDNA) gap accumulation as a potential determinant underlying cisplatin chemosensitivity, at least in some genetic backgrounds, such as BRCA mutations. Cisplatin-induced ssDNA gaps form upon the arrest of replication forks at sites of cisplatin adducts, and restart of DNA synthesis downstream of the lesion through repriming catalyzed by the PRIMPOL enzyme. Here, we show that PRIMPOL overexpression in otherwise wildtype cells results in accumulation of cisplatin-induced ssDNA gaps without sensitizing cells to cisplatin, suggesting that ssDNA gap accumulation does not confer cisplatin sensitivity in BRCA-proficient cells. To understand how ssDNA gaps may cause cellular sensitivity, we employed CRISPR-mediated genome-wide genetic screening to identify factors which enable the cytotoxicity of cisplatin-induced ssDNA gaps. We found that the helicase HELQ specifically suppresses cisplatin sensitivity in PRIMPOL-overexpressing cells, and this is associated with reduced ssDNA accumulation. We moreover identify RAD52 as a mediator of this pathway, and show that RAD52 promotes ssDNA gap accumulation through a BRCA-mediated mechanism. Our work identified the HELQ-RAD52-BRCA axis as a regulator of ssDNA gap processing, shedding light on the mechanisms of cisplatin sensitization in cancer therapy.

Role of Translesion DNA Synthesis in the Metabolism of Replication-associated Nascent Strand Gaps.

Translesion DNA synthesis (TLS) is a DNA damage tolerance pathway utilized by cells to overcome lesions encountered throughout DNA replication. During replication stress, cancer cells show increased dependency on TLS proteins for cellular survival and chemoresistance. TLS proteins have been described to be involved in various DNA repair pathways. One of the major emerging roles of TLS is single-stranded DNA (ssDNA) gap-filling, primarily after the repriming activity of PrimPol upon encountering a lesion. Conversely, suppression of ssDNA gap accumulation by TLS is considered to represent a mechanism for cancer cells to evade the toxicity of chemotherapeutic agents, specifically in BRCA-deficient cells. Thus, TLS inhibition is emerging as a potential treatment regimen for DNA repair-deficient tumors.

Complementary CRISPR genome-wide genetic screens in PARP10-knockout and overexpressing cells identify synthetic interactions for PARP10-mediated cellular survival.

PARP10 is a mono-ADP-ribosyltransferase with multiple cellular functions, including proliferation, apoptosis, metabolism and DNA repair. PARP10 is overexpressed in a significant proportion of tumors, particularly breast and ovarian cancers. Identifying genetic susceptibilities based on PARP10 expression levels is thus potentially relevant for finding new targets for precision oncology. Here, we performed a series of CRISPR genome-wide loss-of-function screens in isogenic control and PARP10-overexpressing or PARP10-knockout cell lines, to identify genetic determinants of PARP10-mediated cellular survival. We found that PARP10-overexpressing cells rely on multiple DNA repair genes for survival, including ATM, the master regulator of the DNA damage checkpoint. Moreover, we show that PARP10 impacts the recruitment of ATM to nascent DNA upon replication stress. Finally, we identify the CDK2-Cyclin E1 complex as essential for proliferation of PARP10-knockout cells. Our work identifies a network of functionally relevant PARP10 synthetic interactions, and reveals a set of factors which can potentially be targeted in personalized cancer therapy.

The TIP60-ATM axis regulates replication fork stability in BRCA-deficient cells.

Maintenance of replication fork stability is essential for genome preservation. Stalled replication forks can be reversed by translocases such as SMARCAL1, and unless protected through the activity of the BRCA pathway, are subsequently subjected to nucleolytic degradation. The ATM and ATR kinases are master regulators of the DNA damage response. ATM activation upon DNA damage is mediated by the acetyltransferase TIP60. Here, we show that the TIP60-ATM pathway promotes replication fork reversal by recruiting SMARCAL1 to stalled forks. This enables fork degradation in BRCA-deficient cells. We also show that this ATM activity is not shared by ATR. Moreover, we performed a series of genome-wide CRISPR knockout genetic screens to identify genetic determinants of the cellular sensitivity to ATM inhibition in wildtype and BRCA2-knockout cells, and validated the top hits from multiple screens. We provide a valuable list of common genes which regulate the response to multiple ATM inhibitors. Importantly, we identify a differential response of wildtype and BRCA2-deficient cells to these inhibitors. In BRCA2-knockout cells, DNA repair genes (including RAD17, MDC1, and USP28) were essential for survival upon ATM inhibitor treatment, which was not the case in wild-type cells. These findings may eventually help guide the way for rational deployment of ATM inhibitors in the clinic.

Comminuted middle third orbito-zygomatic complex fracture leading to blindness due to unanticipated tyre rim explosion during service - A rare case.

An explosion is caused by conversion of solid, liquid into gas with resultant energy release. Blast injuries of large tyres are similar to injuries resulting from landmine explosions. Most of the patients were polytraumatised, initial evaluation and management should follow ATLS. Trauma following tyre blast results in severe soft tissue, orthopedic and head injuries. Head and face is the most commonly affected region followed by upper limb. A 40 year old male patient was watching a car tyre getting inflated with air. Unfortunately the tyre rim exploded on his face, which led to penetrating injury to the eye ball and comminuted middle third fractures. Patient was stabilized and primary hemostasis was achieved. Fractured maxilla was fixed by arch bar wiring and stabilized by using circum-suspension wiring bilaterally. Left eyeball was removed due to open globe injury and intraocular content loss. Unusual maxillofacial injuries are more common. Decision making and treatment of facial penetrating injuries depends on number of factors, which includes location and extent of injury, type of foreign body involved, proximity of vital structures, extent of injury to soft and hard tissue and the relative benefits and risk ratio for the patient. In this case report we have explained about the primary assessment and management of blast injuries.

Seromucinous hamartoma of ethmoid sinus in pediatric patient (case report).

INTRODUCTION AND IMPORTANCE: Seromucinous hamartoma is a rare benign glandular proliferation arising from the respiratory epithelium, which was originally described by Baillie and Batsakis in 1974. Since this time, case reports started to be published on SH, as a middle aged and elderly disease, here we report a case of a pediatric patient who found to have SH. PRESENTATION OF THE CASE: 2-year-old girl, brought by her parent with a complain of a mass at the right medial canthal area for one year. CLINICAL DISCUSSION: As this pediatric patient presented with long standing history of right medial canthal area, we made out differential diagnosis list, with keeping congenital midline nasal masses such as nasal glioma, dermoid, and encephalocele at the top of our differentials, followed by inflammatory disease and lacreimal system disease. After bedside clinical assessment and imaging, patient underwent endoscopic sinus surgery for surgical excision, histopathology analysis came as Seromucinous hamartoma. Postoperative course was unremarkable, patient is disease- free for 18 months, till her most recent follow up. With no additional treatment or recurrence. CONCLUSION: This case report indicates that seromucinous hamartoma should always be considered in the differential diagnosis of pediatric sinonasal disease. According to the literature review we did; this is the first case reported in such an age group.

Assessment of water, sanitation and hygiene service availability in healthcare facilities in the greater Kampala metropolitan area, Uganda.

BACKGROUND: Improved Water, Sanitation and Hygiene (WASH) in Healthcare facilities (HCFs) is of significant public health importance. It is associated with a reduction in the transmission of healthcare acquired infections (HAIs), increased trust and uptake of healthcare services, cost saving from infections averted, increased efficiency and improved staff morale. Despite these benefits, there is limited evidence on availability of WASH services in HCFs in the Greater Kampala Metropolitan Area (GKMA). This study assessed the availability and status of WASH services within HCFs in the GKMA in order to inform policy and WASH programming. METHODS: A cross-sectional study was conducted in 60 HCFs. Availability of WASH services in the study HCFs was assessed using a validated WASH Conditions (WASHCon) tool comprising of structured interviews, HCF observations and microbial water quality analysis. Data were analysed using Stata 14 software and R software. RESULTS: Overall, 84.5% (49/58) and 12.1% (7/58) of HCFs had limited and basic WASH service respectively. About 48.3% (28/58) had limited water service, 84.5% (49/58) had limited sanitation service, 50.0% (29/58) had limited environmental cleanliness service, 56.9% (33/58) had limited hand hygiene service, and 51.7% (30/58) had limited waste management service. About 94.4% of public HCFs had limited WASH service compared to only 68.2% of private not for profit facilities. More health centre IIIs, 92.5% and health centre IVs (85.7%) had limited WASH service compared to hospitals (54.5%). CONCLUSIONS: Our findings indicate that provision of water, sanitation, hand hygiene, environmental cleanliness, and health care waste management services within HCFs is largely hindered by structural and performance limitations. In spite of these limitations, it is evident that environmental cleanliness and treatment of infectious waste can be attained with better oversight and dedicated personnel. Attaining universal WASH coverage in HCFs will require deliberate and strategic investments across the different domains.

Malnutrition amongst HIV adult patients in selected hospitals of Bushenyi district in southwestern Uganda.

BACKGROUND: Malnutrition is an important clinical outcome amongst HIV patients in developing countries and in Uganda, there is scarcity of information on its prevalence and risk factors amongst HIV adult patients. METHODS: A cross-sectional study amongst 253 HIV patients in Bushenyi district assessed their nutritional status using the body mass index (BMI) and mid-upper arm circumference (MUAC), and a questionnaire was used to identify major risk factors. RESULTS: The mean age of the study participants was 38.74 ± 0.80 yrs, while females and males were 52.2% and 47.8% respectively. Prevalence of malnutrition was 10.28% (95% CI: 6.82 - 14.69) in the study. Major socio-economic factors associated with malnutrition were being female, unemployed, dependent and with many family members. Patients with opportunistic infections, low adherence to HAART, and stage of HIV/AIDS had a higher risk of malnutrition. DISCUSSION: In rural communities, a majority of malnourished patients are elderly and these were identified as priority groups for HIV outreach campaigns. The current policy of prioritizing children and women is outdated due to changing disease dynamics, thus showing a need to revise extension service provision in rural communities. CONCLUSIONS: Malnutrition is a threat in HIV adult patients in rural communities of Uganda.

Factors associated with socio-demographic characteristics and antenatal care and iron supplement use in Ethiopia, Kenya, and Senegal.

Antenatal care (ANC) offers remarkable opportunities to reach a large number of women with effective nutrition and health interventions, including iron (Fe) supplementation. However, all women do not equally seek nor benefit from ANC. We aimed to identify characteristics associated with ANC and Fe use among women in hard-to-reach areas in Afar, Ethiopia; Sedhiou and Kolda, Senegal; and Kakamega, Kenya. Women who gave birth within 1 year preceding the survey (n = 4,575) from 15 different sub-regions were randomly selected and surveyed. Multivariable logistic regression was used to identify associations of socio-demographic characteristics with ANC and Fe use. Factors that showed positive associations with ANC uptake included education, income, possession of a mobile phone, and the occupation of the mother or another household member. Beginning ANC in the first trimester associated positively with achievement of 4 or more ANC visits, and having any ANC visits related positively with Fe intake. Distance to the nearest health facility was negatively associated, and type of nearest facility and counselling and health education were positively associated with some outcomes. The results from these surveys demonstrate the need to ensure access of services across all population groups and can help identify ANC programming needs.

Rapid negative inotropic effect induced by TNF-α in rat heart perfused related to PKC activation.

Myocardial depression, frequently observed in septic shock, is mediated by circulating molecules such as cytokines. TNF-α appears to be the most important pro-inflammatory cytokine released during the early phase of a septic shock. It was previously shown that TNF-α had a negative inotropic effect on myocardium. Now, the aim of this study was to investigate the effects of the activation of PKC by TNF-α on heart function, and to determine if this cytokine could induce a decrease of membrane excitability. Isolated rat hearts (n = 6) were perfused with Tyrode solution containing TNF-α at 20 ng/ml during 30 min by using a Langendorff technique. Expressions of PKC-α and PKC-ε were analysed by western blot on membrane and cytosol proteins extracted from ventricular myocardium. Patch clamp was performed on freshly isolated cardiomyocytes (n = 8). Compared to control situation, 30 min of TNF-α perfusion led to cardiac dysfunction with a decrease of the heart rate (-83%), the force (-20%) and speed of relaxation (-18%) and the coronary flow (-25%). This is associated with an activation and a membrane targeting of both PKC-α and PKC-ε isoforms in ventricle with respectively +123% and +54% compared to control hearts. Nevertheless, TNF-α had no significant effect on voltage-gated sodium current (109.0%+/- 12.5) after addition of the cytokine when compared to control. These results showed that TNF-α had a negative inotropic effect on the isolated rat heart and can induce PKC activation leading to an impaired contractility of the heart. However the early heart dysfunction induced by the cytokine was not associated to a decrease of cardiomyocytes membrane excitability as it has been evidenced in skeletal muscle fibres.

Saturation Dynamics Measures Absolute Cross Section and Generates Contrast within a Neuron.

The intensity required to optically saturate a chromophore is a molecular property that is determined by its absorption cross section (σ) and the excited state lifetime. We present an analytical description of such a system and show that fluorescence around the onset of saturation is characterized by product of absorption cross section and lifetime. Using this approach we formulate a generalized method for measuring the multiphoton cross section of fluorophores and use it to obtain the absolute three-photon cross-section spectra of tryptophan. We find that the tryptophan three-photon cross section ranges from 0.28 S.I. units (m(6)s(2)photon(-2)) at 870 nm to 20 S.I. units at 740 nm. Further, we show that the product of molecular rate of excitation and de-excitation, denoted as ß, serves as a vital contrasting agent for imaging local environment. Our contrast parameter, ß, is related to fraction of the population present in the excited state and is independent of the fluorophore concentration. We show that ß-imaging can be carried out in a regular two-photon microscope setup through a series of intensity scans. Using enhanced green fluorescent protein (EGFP) fluorescence from the brain slices of Thy-1 EGFP transgenic mice, we show that there is an inherent, concentration independent, variation in contrast across the soma and the dendrite.

A Comparative Study Between Bupivacaine with Adrenaline and Carbonated Bupivacaine with Adrenaline for Surgical Removal of Impacted Mandibular Third Molar.

OBJECTIVES: To compare the effectiveness of bupivacaine with adrenaline with that of carbonated bupivacaine with adrenaline on pain, onset of anesthesia and duration of anesthesia following surgical removal of impacted mandibular third molar. STUDY DESIGN: All the patients who underwent surgical removal of impacted mandibular third molar and who fulfilled our inclusion and exclusion criteria from 1st June 2013 to 30th June 2014 were included in our study. Patients who were diagnosed as having impacted mandibular third molar were randomly allocated to two groups namely group A (bupivacaine with adrenaline), group B (carbonated bupivacaine with adrenaline). Pain during deposition of local anesthetic, onset of anesthesia and duration of anesthesia were compared between the two groups. The collected data were subjected to statistical analysis by Chi Square test, Mann-Whitney U test. RESULTS AND CONCLUSION: The efficacy of carbonated bupivacaine with adrenaline is more compared with bupivacaine with adrenaline in decreasing pain on deposition of local anesthetic solution and in rapid onset of anesthesia. The duration of anesthesia for carbonated bupivacaine with adrenaline and bupivacaine with adrenaline had no significant difference. The use of carbonated bupivacaine with adrenaline will reduce the patient discomfort both intra-operatively and post-operatively.

The neuron-derived orphan receptor 1 (NOR1) is induced upon human alternative macrophage polarization and stimulates the expression of markers of the M2 phenotype.

BACKGROUND: Atherosclerosis is an inflammatory disease in which macrophages play a crucial role. Macrophages are present in different phenotypes, with at the extremes of the spectrum the classical M1 pro-inflammatory and the alternative M2 anti-inflammatory macrophages. The neuron-derived orphan receptor 1 (NOR1), together with Nur77 and Nurr1, are members of the NR4A orphan nuclear receptor family, expressed in human atherosclerotic lesion macrophages. However, the role of NOR1 in human macrophages has not been studied yet. OBJECTIVES: To determine the expression and the functions of NOR1 in human alternative macrophages. METHODS AND RESULTS: In vitro IL-4 polarization of primary monocytes into alternative M2 macrophages enhances NOR1 expression in human but not in mouse macrophages. Moreover, NOR1 expression is most abundant in CD68+MR+ alternative macrophage-enriched areas of human atherosclerotic plaques in vivo. Silencing NOR1 in human alternative macrophages decreases the expression of several M2 markers such as the Mannose Receptor (MR), Interleukin-1 Receptor antagonist (IL-1Ra), CD200 Receptor (CD200R), coagulation factor XIII A1 polypeptide (F13A1), Interleukin 10 (IL-10) and the Peroxisome Proliferator-Activated Receptor (PPAR)γ. Bioinformatical analysis identified F13A1, IL-1Ra, IL-10 and the Matrix Metalloproteinase-9 (MMP9) as potential target genes of NOR1 in human alternative macrophages. Moreover, expression and enzymatic activity of MMP9 are induced by silencing and repressed by NOR1 overexpression in M2 macrophages. CONCLUSIONS: These data identify NOR1 as a transcription factor induced during alternative differentiation of human macrophages and demonstrate that NOR1 modifies the alternative macrophage phenotype.

The coronary artery disease-associated gene C6ORF105 is expressed in human macrophages under the transcriptional control of PPARγ.

Coronary artery disease (CAD) is a major cause of morbidity and mortality. Mutations in C6ORF105, associated with decreased gene expression, positively correlate with the risk of CAD in Chinese populations. Moreover, the C6ORF105-encoded protein may play a role in coagulation. Here, we report that C6ORF105 gene expression is lower in circulating mononuclear cells from obese diabetic than lean subjects. Moreover, C6ORF105 is expressed in human macrophages and atherosclerotic lesions, where its expression positively correlates with expression of the transcription factor Peroxisome Proliferator-Activated Receptor (PPAR)γ. Activation of PPARγ increases, in a PPARγ-dependent manner, the expression of C6ORF105 in human macrophages and atherosclerotic lesions.

[Thrombocytosis in patients treated with low-molecular-weight heparin: more common than imagined?]. / La thrombocytose au cours des traitements par héparine de bas poids moléculaire : une situation plus fréquente qu'on ne le croit ?

BACKGROUND: Use of low-molecular-weight heparin (LMWH) is widespread. Clinicians are well aware of LMWH-related thrombopenia, but reports of thrombocytosis are more exceptional. We evaluated prospectively the incidence of thrombocytosis (>450 G/L) in the general medicine setting among patients needing LMWH treatment. PATIENTS AND METHODS: We followed for the duration of treatment 95 consecutive patients receiving LMWH and managed in a general medicine setting. Thrombotic events, bleeding and platelet counts were noted. RESULTS: Among the 95 patients, 29 developed thrombocytosis during the follow-up (587±102 G/L). In 15 patients, thrombocytosis occurred early after discharge ; in 14 others the counts rose to a pathological level on average 5.4±0.7 days after discharge then returned to normal levels spontaneously in 10.7±7.9 days. Only one clinical event (erysipelas) was reported, potentially associated with this thrombocytosis. There were no thrombotic or hemorrhagic events during the follow-up. DISCUSSION: In our population, the observed cases of thrombocytosis were moderate. Incidence was however not exceptional despite the absence of any notable adverse event, in agreement with the rare data in the literature.

Drug-eluting stents: trading restenosis for thrombosis?

Within the last 6 years, it has been demonstrated that drug-eluting stents (DES) reduce significantly angiographic and clinical restenosis after percutaneous coronary interventions. These results are consistent across several clinical randomized controlled trials comparing these new devices with bare metallic stents (BMS), which themselves have already markedly improved the results obtained with balloon angioplasty in the early days of this method of myocardial revascularization. Nevertheless, some concerns have been raised regarding a delayed endothelialization of the coated prostheses leading to late stent thrombosis occurring mainly when antiplatelet therapy is discontinued in the follow-up. The most recent data show that, in comparison with BMS, there is a small excess of late (> 1 year) stent thrombosis but this is not associated with an increased risk of death or myocardial infarction or all cause mortality. These concerns do not outweigh the strong benefits of DES in preventing restenosis but require a number of measures concerning a longer dual antiplatelet treatment (than initially expected), to control patient treatment compliance and to provide a complete education of patients and physicians. Future devices dealing with the two issues (antiproliferative properties with rapid controlled endothelialization preventing thrombosis) would be the next major advance in this rapidly evolving field.

[An experimental model of pulmonary valve implantation: a percutaneous and transventricular approach without cardiopulmonary bypass]. / Modèle de mise en place d'une valve pulmonaire: approche percutanée puis trans-ventriculaire sans circulation extracorporelle.

Pulmonary valve replacement by a catheter procedure remains a therapeutic challenge. In this report, the authors demonstrate the possibility of implantation of a porcine xenograft specially prepared on an auto-expanding stent (valved stent) in a sheep model. The porcine xenograft was prepared with hypotonic non-enzymatic solutions. It was sewn onto an auto-expanding stent (Luminex Bard) and inserted into an introduction sheath of 22-24 F (Gore) calibre. In a preliminary approach, the catheter was inserted through the jugular vein. Out of 6 attempts, it was possible to position the valved stent in the pulmonary position in two cases but all the animals died of different causes: tamponade, arrhythmias, air embolism. Following this experience, two valves were implanted through the superior and inferior vena cavae. This first percutaneous approach has been modified to a mixed medico-surgical approach with a transventricular introduction without cardiopulmonary bypass. This was performed through a left thoracotomy with puncture of the pulmonary infundibulum using the same systems of introduction and valved stent. Three implantations were successfully performed. In addition, a reduction of the size of the pulmonary artery was realised to prevent embolisation of the valved stent to the pulmonary artery or one of its branches. The transventricular approach is feasible for implantation of pulmonary valve prosthesis on a stent. This technique could be adapted for correction of pulmonary regurgitation after correction of Tetralogy of Fallot associated with reduction of the pulmonary infundibulum.

Relevance of tissue factor in cardiovascular disease.

Overexpression and exposition of tissue factor (TF) in atherosclerotic plaques and/or arterial thrombi are critical events in atherothrombosis. TF, the receptor for factor VII (FVII) and activated factor VII (FVIIa), is the principal initiator of blood coagulation and induces thrombin generation leading to fibrin formation and platelet activation. TF also plays a major role in cell migration and angiogenesis. TF activity is downregulated by Tissue Factor Pathway Inhibitor (TFPI), a Kunitz-type inhibitor, which forms a neutralizing complex with TF, FVIIa and activated factor X. In physiological conditions, TF is absent from vascular cells which come into contact with flowing blood and is present as an inactive pool in fibroblasts and smooth muscle cells (SMC). In contrast, TF is widely expressed in atherosclerotic plaques and is found in macrophages, SMCs, and foam-cells and also in extracellular matrix and acellular lipid-rich core. TF expression is up-regulated by inflammatory cytokines and oxidized lipids. Plaque thrombogenicity is directly correlated to their TF content. After fibrous cap disruption, TF is exposed on plaque surface and triggers thrombus formation leading to arterial lumen occlusion and/or downstream embolization. In coronary and carotid plaques, TF content was found to be higher in plaques from symptomatic than asymptomatic patients. Soluble forms of TF and microparticles of monocyte and platelet origin, and bearing TF, constitute "blood-born TF". The contribution of this TF pool to arterial thrombosis is still under discussion. TF pathway is a target for new therapeutic agents that can decrease TF activity, such as active site-inactivated factor VIIa, recombinant TFPI and antibodies against TF or peptides interfering with TF-FVIIa complex activity.