Human Clinical Samples Research Articles

Tetrahydrocurcumin targets TRIP13 inhibiting the interaction of TRIP13/USP7/c-FLIP to mediate c-FLIP ubiquitination in triple-negative breast cancer

IntroductionTriple-negative breast cancer (TNBC) has a high mortality rate and limited treatment options. Tetrahydrocurcumin (THC), a major metabolite of curcumin, has potential antitumor activities. However, the antitumor effects and mechanism of THC in TNBC remain elusive. ObjectivesTo investigate the mechanism of THC in combating TNBC by targeting TRIP13 to disrupt the interaction of the TRIP13/USP7/c-FLIP complex and mediate c-FLIP ubiquitination both in vitro and in vivo. MethodsApoptosis was measured by TUNEL and flow cytometry. Click chemistry-based target fishing, CETSA, DARTS, and SPR were used to identify direct target of THC. Protein interactions was examined using co-immunoprecipitation. The role of USP7 in THC-mediated c-FLIP ubiquitination was evaluated by in vitro deubiquitination assay. Human breast cancer clinical samples were employed to assess the expression of c-FLIP, TRIP13, and USP7. The impact of THC on USP7/TRIP13/c-FLIP was analyzed using co-immunoprecipitation, confocal microscopy, molecular docking and dynamics simulations. ResultsTHC effectively inhibits TNBC cell proliferation and tumor growth in vitro and in vivo without significant toxicity. Mechanistically, THC induces extrinsic apoptosis in TNBC primarily by promoting degradation of c-FLIP, a key negative regulator in the apoptotic pathway. Furthermore, utilizing click chemistry-based target fishing, we identified TRIP13, a component of the highly conserved AAA ATPase family, as a direct target of THC in combating TNBC. Interestingly, contrary to previous drug-target studies, the knockdown of TRIP13 further amplified the antitumor effects of THC. After in-depth investigation, it was revealed that TRIP13 forms a trimeric complex with USP7 and c-FLIP in TNBC cells. THC specifically targets TRIP13 to disrupt the interaction of TRIP13/USP7/c-FLIP, leading to the ubiquitination of c-FLIP, ultimately inducing extrinsic apoptosis. ConclusionsThese findings offer new insights into the novel molecular mechanisms of anti-TNBC effects of THC and present a promising targeted therapeutic strategy for TNBC.

Comparative Phenotypic and Genotypic Properties of Serratia marcescens Isolates from Clinical Cases from human in AL-Diwaniyah Province, Iraq

Serratia marcescens is a Gram-negative, rod-shaped bacterium. It is an opportunistic and facultative anaerobe. This study aims to determine the occurrence rates of Serratia marcescens in human clinical samples, investigate the pigM gene, sequence the isolates, and compare them with global isolates documented in the NCBI database. Fifty burn samples were collected from the specialized center for burns in AL-Diwaniyah province, along with 35 urine samples from individuals suffering from UTIs from Al-Diwaniyah Teaching Hospital and various private laboratories. These samples were transferred in a cooled container to the microbiology lab at the Veterinary Medicine College, University of Al-Qadisiyah, for bacteriological assay. The samples were then cultured using selective and differential media. Following the purification of the cultured bacteria, biochemical tests were conducted to confirm the isolates. Results showed that 10 out of 85 samples (11.76%) were positive for Serratia marcescens. Culture positivity was most frequent in urine samples (17.1%), followed by burn samples (8%). Molecular methods revealed that human isolates gave 100% positive results for 16S rRNA. The study found that ten isolates of Serratia marcescens possess the pigM gene, with the 16S rRNA gene sequenced using the BLASTN algorithm of NCBI. All isolates were recorded under accession numbers (OR835629, OR835630, OR835631, OR835632, OR835633, OR835634, OR835635, OR835636, OR835637 and OR835638, showing 97-99% identity with isolates detected in China, India, and Nigeria. It can be concluded that the isolation rates of Serratia marcescens from urine samples are higher than those from burn samples. The sequencing results showed a 97-99% similarity with global isolates from China, India, and Nigeria.

Molecular Patterns and Antimicrobial Resistance Characterization of Salmonella enterica Non-Typhoidal from Human, Food, and Environment Samples Isolated in Luanda, Angola

The aim of this study was to characterize the antimicrobial resistance phenotype and genotype of non-typhoidal Salmonella spp. isolated in Luanda, Angola. Between 2013 and 2015, human clinical samples, food, and environmental samples (n = 290) were collected at different regions of Luanda city and screened for the presence of Salmonella spp. Bacterial isolates were preliminarily identified using the API 20E Kit, and their identification was confirmed using PCR and serotyping. All Salmonella spp. isolates were tested by minimum inhibitory concentration against 19 antimicrobials. The isolates were also screened using PCR for the presence of resistance genes (blaOXA-1, blaSHV, blaTEM, sul1, sul2, sul3, qnrA, qnrB, qnrS, qnrC, qnrD, aac(6′)-Ib, dfrIa [targeting dfrA1, dfrA5, dfrA15, dfrA15b, dfrA16, dfrA16b] and dfrA12, cmlA, and floR) and typed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Salmonella enterica non-typhoidal was detected in 21.3% of the clinical samples (n = 32/150), 11.1% of the food samples (n = 10/90), and 26% of the environmental samples (n = 13/50). Serotyping revealed that the monophasic variant of Salmonella Typhimurium (Salmonella enterica serovar 4,[5],12:i:-) was detected in 38.1% of the samples. Moreover, serovar Salmonella Enteritidis was the second most frequent. Only 7.3% of the isolates were resistant to at least one antimicrobial. Furthermore, isolates from different origins (clinical, environmental, and food) were associated with the same lineages, Salmonella Enteritidis ST11 and S. enterica ser. Typhimurium ST313. The detection of S. enterica serovar 4,[5],12:i:- in different settings reinforces the need for a One Health approach to control this zoonosis in Angola.

DHA plays a protective role in cerebral ischemia–reperfusion injury by affecting macrophage/microglia type polarization

A close correlation exists between the macrophage/microglia(MΦ/MG) polarization states and the development of cerebral ischemia and reperfusion (I/R). Therefore it is of great significance to research on how to modulate the MΦ/MG states for improved patient outcomes. In particular, regulatory mechanisms involved in this process remain to be identified. Hereby, we aim to shed light on how docosahexaenoic acid (DHA) actively modulates the switch between M1 and M2 macrophage states by restraining the NACHT-LRR-PYD-containing protein three inflammasome (NALP3). We found that NALP3-positive cells were detected in clinical human cerebral infarction tissue samples and the mouse tMCAO model. In mice after DHA treatment, the number of NALP3-positive cells was significantly reduced, significantly decreasing infarct volume and improving the postoperative physical status of mice. NALP3-positive cells were found to be MΦ/MG after co-staining with CD11b. By extracting peritoneal macrophages, it was verified that DHA inhibited the activation of NALP3 and regulated the transformation of M1 and M2 cells, thereby reducing I/R injury.

Morusin, a novel inhibitor of ACLY, induces mitochondrial apoptosis in hepatocellular carcinoma cells through ROS-mediated mitophagy

ObjectiveMorusin (Mor), a prenylated flavonoid isolated from the root bark of Morus alba L., exhibits potent anti-tumour effects; however, the molecular target of Mor is still not entirely clear. This study aimed to elucidate the mechanism of Mor against hepatocellular carcinoma (HCC) and identify potential molecular targets. MethodsMitochondrial function was assessed by measuring the mitochondrial membrane potential, mitochondrial ultrastructure, oxygen consumption, and ATP levels. Mor-induced mitophagy was confirmed using western blotting, immunofluorescence, and fluorescent probes. Transcriptomics, flow cytometry, western blotting, qRT-PCR and biochemical assays were used to reveal the molecular mechanisms and targets of Mor against HCC. We further validated the interaction between Mor and the target proteins using molecular docking and biolayer interferometry (BLI). The inhibitory effect of Mor in vivo was evaluated using a Hep3B murine xenograft model. ResultsMor significantly reduced the ATP citrate lyase (ACLY) expression and inhibited ACLY activity in HCC cells. BLI analysis demonstrated a direct interaction between Mor and the ACLY active domain. Mor-induced ACLY inhibition led to ROS accumulation in HCC cells, which caused mitochondrial damage, triggered PINK1/Parkin-mediated mitophagy, and ultimately induced mitochondrial apoptosis. We further verified that ROS is crucial in the apoptotic action of Mor through experiments regarding an ROS scavenger. Mor also significantly inhibited tumour xenograft growth in vivo. In addition, analysis of human liver cancer clinical samples revealed elevated ACLY levels positively correlated with histologic grade. ConclusionCollectively, our findings highlight Mor as a potent bioactive inhibitor of ACLY and a promising candidate for HCC therapy.

Evaluation of an in-house pan-Malassezia quantitative PCR in human clinical samples.

Althought Malassezia spp. have been involved in various pathologies, they are an integral part of the cutaneous, gut, oral, ears, nose and throat (ENT) mycobiota. Since Malassezia are difficult to grow in culture, unexhaustive molecular biology methods have been developed to detect them. The aim of the study was to evaluate an in-house pan-Malassezia quantitative polymerase chain reaction (panM-qPCR) on various clinical human samples and determine Malassezia burden in various human mycobiota. The panM-qPCR was designed to target the repeated 28S rDNA gene from all Malassezia species. We used the assay to quantify the Malassezia burden on 361 samples from 161 subjects (80 skin swabs from 10 healthy volunteers (HV), 13 samples from 2 seborrheic dermatitis patients (SD), 90 skin samples from 19 burned patients, 119 stool samples from 89 immunocompromised patients, 59 ENT samples from 41 patients). For HV, the amount of Malassezia was different according to the swabbed areas. Quantification cycle (Cq) in SD is lower than in HV. In burned patients, Cq was significantly lower compared to HV. In stool samples, 6.7% were positive for Malassezia spp. with a high Cq. For the ENT area, a higher proportion of positive specimens were detected in ear samples than in nose samples. Our findings emphasized the importance of qPCR, confirming elevated Malassezia spp. levels on individuals' faces and scalps, increased burden in SD patients and in severely burnt patients than in HV. The pan-MqPCR appears to be a promising tool for studying Malassezia in various human mycobiota.

Long-term persistence of diverse clones shapes the transmission landscape of invasive Listeria monocytogenes

BackgroundThe foodborne bacterium Listeria monocytogenes (Lm) causes a range of diseases, from mild gastroenteritis to invasive infections that have high fatality rate in vulnerable individuals. Understanding the population genomic structure of invasive Lm is critical to informing public health interventions and infection control policies that will be most effective especially in local and regional communities.MethodsWe sequenced the whole draft genomes of 936 Lm isolates from human clinical samples obtained in a two-decade active surveillance program across 58 counties in New York State, USA. Samples came mostly from blood and cerebrospinal fluid. We characterized the phylogenetic relationships, population structure, antimicrobial resistance genes, virulence genes, and mobile genetic elements.ResultsThe population is genetically heterogenous, consisting of lineages I–IV, 89 clonal complexes, 200 sequence types, and six known serogroups. In addition to intrinsic antimicrobial resistance genes (fosX, lin, norB, and sul), other resistance genes tetM, tetS, ermG, msrD, and mefA were sparsely distributed in the population. Within each lineage, we identified clusters of isolates with ≤ 20 single nucleotide polymorphisms in the core genome alignment. These clusters may represent isolates that share a most recent common ancestor, e.g., they are derived from the same contamination source or demonstrate evidence of transmission or outbreak. We identified 38 epidemiologically linked clusters of isolates, confirming eight previously reported disease outbreaks and the discovery of cryptic outbreaks and undetected chains of transmission, even in the rarely reported Lm lineage III (ST3171). The presence of animal-associated lineages III and IV may suggest a possible spillover of animal-restricted strains to humans. Many transmissible clones persisted over several years and traversed distant sites across the state.ConclusionsOur findings revealed the bacterial determinants of invasive listeriosis, driven mainly by the diversity of locally circulating lineages, intrinsic and mobile antimicrobial resistance and virulence genes, and persistence across geographical and temporal scales. Our findings will inform public health efforts to reduce the burden of invasive listeriosis, including the design of food safety measures, source traceback, and outbreak detection.

Anticancer agent 5-fluorouracil reverses meropenem resistance in carbapenem-resistant Gram-negative pathogens

The global increasing incidence of clinical infections caused by carbapenem-resistant Gram-negative pathogens requires urgent and effective treatment strategies. Antibiotic adjuvants represent a promising approach to enhance the efficacy of meropenem against carbapenem-resistant bacteria. This study shows that the anticancer agent 5-fluorouracil (5-FU, 50 µM) significantly reduced the minimum inhibitory concentration of meropenem against blaNDM-5 positive Escherichia coli by 32-fold through cell-based high-throughput screening. Further pharmacological studies indicated that 5-FU exhibited potentiation effects on carbapenem antibiotics against 42 Gram-negative bacteria producing either metallo-β-lactamases (MBLs), such as NDM and IMP, or serine β-lactamases (Ser-BLs), like KPC and OXA. These bacteria included E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp., 32 of which were obtained from human clinical samples. Mechanistic investigations revealed that 5-FU inhibited the transcription and expression of the blaNDM-5 gene. In addition, 5-FU combined with meropenem enhanced bacterial metabolism, and stimulated the production of reactive oxygen species (ROS), thereby rendering bacteria more susceptible to meropenem. In a mouse systemic infection model, 5-FU combined with meropenem reduced bacterial loads and effectively elevated the survival rate of 83.3%, compared with 16.7% with meropenem monotherapy. Collectively, these findings indicate the potential of 5-FU as a novel meropenem adjuvant to improve treatment outcomes against infections caused by carbapenem-resistant bacteria.

YTHDF1 regulates GID8-mediated glutamine metabolism to promote colorectal cancer progression in m6A-dependent manner

Dysregulation of epigenetics is a hallmark of cancer development, and YTHDF1 stands out as a crucial epigenetic regulator with the highest DNA copy number variation among all N6-methyladenosine (m6A) regulators in colorectal cancer (CRC) patients. Here, we aimed to investigate the specific contribution of YTHDF1 overexpression to CRC progression and its consequences. Through multiple bioinformatic analyses of human cancer databases and clinical CRC samples, we identified GID8/Twa1 as a crucial downstream target of YTHDF1. YTHDF1 manipulates GID8 translation efficiency in an m6A-dependent manner, and high expression of GID8 is associated with more aggressive tumor progression and poor overall survival. Mechanistically, GID8 is intimately associated with glutamine metabolic demands by maintaining active glutamine uptake and metabolism through the regulation of excitatory amino acid transporter 1 (SLC1A3) and glutaminase (GLS), thereby facilitating the malignant progression of CRC. Inhibition of GID8 attenuated CRC proliferation and metastasis both in vitro and in vivo. In summary, we identified a previously unknown target pertaining to glutamine uptake and metabolism in tumor cells, underscoring the potential of GID8 in the treatment of CRC.

Comparative genomic analysis of an emerging Pseudomonadaceae member, Thiopseudomonas alkaliphila.

Thiopseudomonas alkaliphila, an organism recently classified within the Pseudomonadaceae family, has been detected in diverse sources such as human tissues, animal guts, industrial fermenters, and decomposition environments, suggesting a diverse ecological role. However, a large knowledge gap exists in how T. alkaliphila functions. In this comparative genomic analysis, adaptations indicative of habitat specificity among strains and genomic similarity to known opportunistic pathogens are revealed. Genomic investigation reveals a core metabolic utilization of multiple oxidative and non-oxidative catabolic pathways, suggesting adaptability to varied environments and carbon sources. The genomic repertoire of T. alkaliphila includes secondary metabolites, such as antimicrobials and siderophores, indicative of its involvement in microbial competition and resource acquisition. Additionally, the presence of transposases, prophages, plasmids, and Clustered Regularly Interspaced Short Palindromic Repeats-Cas systems in T. alkaliphila genomes suggests mechanisms for horizontal gene transfer and defense against viral predation. This comprehensive genomic analysis expands our understanding on the ecological functions, community interactions, and potential virulence of T. alkaliphila, while emphasizing its adaptability and diverse capabilities across environmental and host-associated ecosystems.IMPORTANCEAs the microbial world continues to be explored, new organisms will emerge with beneficial and/or pathogenetic impact. Thiopseudomonas alkaliphila is a species originally isolated from clinical human tissue and fluid samples but has not been attributed to disease. Since its classification, T. alkaliphila has been found in animal guts, animal waste, decomposing remains, and biogas fermentation reactors. This is the first study to provide an in-depth view of the metabolic potential of publicly available genomes belonging to this species through a comparative genomics and draft pangenome calculation approach. It was found that T. alkaliphila is metabolically versatile and likely adapts to diverse energy sources and environments, which may make it useful for bioremediation and in industrial settings. A range of virulence factors and antibiotic resistances were also detected, suggesting T. alkaliphila may operate as an undescribed opportunistic pathogen.

Some virulence genes are associated with antibiotic susceptibility in Enterobacter cloacae complex

BackgroundEnterobacter cloacae complex (ECC) including different species are isolated from different human clinical samples. ECC is armed by many different virulence genes (VGs) and they were also classified among ESKAPE group by WHO recently. The present study was designed to find probable association between VGs and antibiotic susceptibility in different ECC species.MethodsForty-five Enterobacter isolates that were harvested from different clinical samples were classified in four different species. Seven VGs were screened by PCR technique and antibiotic susceptibility assessment was performed by disk-diffusion assay.ResultFour Enterobacter species; Enterobacter cloacae (33.3%), Enterobacter hormaechei (55.6%), Enterobacter kobei (6.7%) and Enterobacter roggenkampii (4.4%) were detected. Minimum antibiotic resistance was against carbapenem agents and amikacin even in MDR isolates. 33.3% and 13.3% of isolates were MDR and XDR respectively. The rpoS (97.8%) and csgD (11.1%) showed maximum and minimum frequency respectively. Blood sample isolated were highly virulent but less resistant in comparison to the other sample isolates. The csgA, csgD and iutA genes were associated with cefepime sensitivity.ConclusionThe fepA showed a predictory role for differentiating of E. hormaechei from other species. More evolved iron acquisition system in E. hormaechei was hypothesized. The fepA gene introduced as a suitable target for designing novel anti-virulence/antibiotic agents against E. hormaechei. Complementary studies on other VGs and ARGs and with bigger study population is recommended.

Using FASS-LTP in postmortem mice brain tissues to assess pathological synaptic function

BackgroundStudy of synaptic integrity using conventional electrophysiology is a gold standard for quantitative assessment of neurodegeneration. Fluorescence assisted single-synapse long-term potentiation (FASS-LTP) provides a high throughput method to assess the synaptic integrity of neurotransmission within and between different brain regions as a measure of pharmacological efficacy in translational models. New methodWe adapted the existing method to our purpose by adding a step during the thawing of frozen samples, by an extra step of placing them on a rocker at room temperature for 30 minutes immediately following thawing with constant mixing on a shaker. This allowed for gradual, uniform thawing, effectively separating the synaptosomes. Our study demonstrates FASS-LTP on four brain regions at 6- and 12-month periods in the 3xTg-AD mouse model, treating sibling cohorts with VU0155069 (a small molecule inhibitor) or vehicle (0.9 % saline). ResultsOur findings demonstrate the robust ability of the FASS-LTP technique to characterize the functional synaptic integrity maintained by disease-treatment therapies in multiple brain regions longitudinally using frozen brain tissue. Comparison with existing methodsBy providing a detailed, user-friendly protocol for this well-known analysis and including a recovery step improved the ability to robustly replicate the FASS-LTP between different brain regions. This may be extrapolated to a translational use on human clinical samples to improve understanding of the therapeutic impact on synaptic performance related to glutamate neurotransmission. ConclusionsFASS-LTP method offers a robust analysis of synaptosomes isolated from frozen tissue samples, demonstrating greater reproducibility in rodent and human synapses in physiological and pathological states.

RNF31-mediated IKKα ubiquitination aggravates inflammation and intestinal injury through regulating NF-κB activation in human and mouse neonates

AimsNeonatal necrotizing enterocolitis (NEC) is a leading cause of intestine inflammatory disease, and macrophage is significantly activated during NEC development. Posttranslational modifications (PTMs) of proteins, particularly ubiquitination, play critical roles in immune response. This study aimed to investigate the effects of ubiquitin-modified proteins on macrophage activation and NEC, and discover novel NEC-related inflammatory proteins. Materials and methodsProteomic and ubiquitin proteomic analyses of intestinal macrophages in NEC/healthy mouse pups were carried out. In vitro macrophage inflammation model and in vivo NEC mouse model, as well as clinical human samples were used for further verification the inhibitor of nuclear factor-κB kinase α (IKKα) ubiquitination on NEC development through Western blot, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry. Key findingsWe report here that IKKα was a new ubiquitin-modified protein during NEC through ubiquitin proteomics, and RING finger protein 31 (RNF31) acted as an E3 ligase to be involved in IKKα degradation. Inhibition of IKKα ubiquitination and degradation with siRNF31 or proteasome inhibitor decreased nuclear factor-κB (NF-κB) activation, thereby decreasing the expression of pro-inflammatory factors and M1 macrophage polarization, resulting in reliving the severity of NEC. SignificanceOur study suggests the activation of RNF31-IKKα-NF-κB axis triggering NEC development and suppressing RNF31-mediated IKKα degradation may be therapeutic strategies to be developed for NEC treatment.

Interaction of the tumor suppressor SMAD4 and WNT signaling in progression to oral squamous cell carcinoma.

SMAD4 is a tumor suppressor mutated or silenced in multiple cancers, including oral cavity squamous cell carcinoma (OSCC). Human clinical samples and cell lines, mouse models and organoid culture were used to investigate the role that SMAD4 plays in progression from benign disease to invasive OSCC. Human OSCC lost detectable SMAD4 protein within tumor epithelium in 24% of cases, and this loss correlated with worse progression-free survival independent of other major clinical and pathological features. A mouse model engineered for KrasG12D expression in the adult oral epithelium induced benign papillomas, however the combination of KrasG12D with loss of epithelial Smad4 expression resulted in rapid development of invasive carcinoma with features of human OSCC. Examination of regulatory pathways in 3D organoid cultures of SMAD4+ and SMAD4- mouse tumors with Kras mutation found that either loss of SMAD4 or inhibition of TGFβ signaling upregulated the WNT pathway and altered the extracellular matrix. The gene signature of the mouse tumor organoids lacking SMAD4 was highly similar to the gene signature of human head and neck squamous cell carcinoma. In summary, this work has uncovered novel mechanisms by which SMAD4 acts as a tumor suppressor in OSCC. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Isophorone-based crystallization-induced-emission sensors detect proteome aggregation in live cells and tissues with breast cancer

BackgroundProtein misfolding and aggregation can lead to various diseases. Recent studies have shed light on the aggregated protein in breast cancer pathology, which suggests that it is crucial to design chemical sensors that visualize protein aggregates in breast cancer, especially in clinical patient-derived samples. However, most reported sensors are constrained in cultured cell lines. ResultsIn this work, we present the development of two isophorone-based crystallization-induced-emission fluorophores for detecting proteome aggregation in breast cancer cell line and tissues biopsied from diseased patients, designated as A1 and A2. These probes exhibited viscosity sensitivity and recovered their fluorescence strongly at crystalline state. Moreover, A1 and A2 exhibit selective binding capacity and strong fluorescence for various aggregated proteins. Utilizing these probes, we detect protein aggregation in stressed breast cancer cells, xenograft mouse model of human breast cancer and clinical patient-derived samples. Notably, the fluorescence intensity of both probes light up in tumor tissues. SignificanceThe synthesized isophorone-based crystallization-induced-emission fluorophores, A1 and A2, enable sensitive detection of protein aggregation in breast cancer cells and tissues. In the future, aggregated proteins are expected to become indicators for early diagnosis and clinical disease monitoring of breast cancer.

PAK4 inhibition augments anti-tumour effect by immunomodulation in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumours, warranting novel treatments. Here, we examined the therapeutic efficacy of inhibiting p21 activated kinase 4 (PAK4) in OSCC and determined its immunomodulatory effect by focusing on the enhancement of anti-tumour effects. We examined PAK4 expression in OSCC cells and human clinical samples and analysed the proliferation and apoptosis of OSCC cells following PAK4 inhibition in vitro. We also investigated the effects of in vivo administration of a PAK4 inhibitor on immune cell distribution and T-cell immune responses in OSCC tumour-bearing mice. PAK4 was detected in all OSCC cells and OSCC tissue samples. PAK4 inhibitor reduced the proliferation of OSCC cells and induced apoptosis. PAK4 inhibitor significantly attenuated tumour growth in mouse and was associated with increased proportions of IFN-γ-producing CD8+ T-cells. Furthermore, PAK4 inhibitor increased the number of dendritic cells (DCs) and up-regulated the surface expression of various lymphocyte co-stimulatory molecules, including MHC-class I molecules, CD80, CD83, CD86, and CD40. These DCs augmented CD8+ T-cell activation upon co-culture. Our results suggest that PAK4 inhibition in OSCC can have direct anti-tumour and immunomodulatory effects, which might benefit the treatment of this malignancy.

Improved Mass Spectrometry–Based Methods Reveal Abundant Propionylation and Tissue-Specific Histone Propionylation Profiles

Histone posttranslational modifications (PTMs) have crucial roles in a multitude of cellular processes, and their aberrant levels have been linked with numerous diseases, including cancer. Although histone PTM investigations have focused so far on methylations and acetylations, alternative long-chain acylations emerged as new dimension, as they are linked to cellular metabolic states and affect gene expression through mechanisms distinct from those regulated by acetylation. Mass spectrometry is the most powerful, comprehensive, and unbiased method to study histone PTMs. However, typical mass spectrometry–based protocols for histone PTM analysis do not allow the identification of naturally occurring propionylation and butyrylation. Here, we present improved state-of-the-art sample preparation and analysis protocols to quantitate these classes of modifications. After testing different derivatization methods coupled to protease digestion, we profiled common histone PTMs and histone acylations in seven mouse tissues and human normal and tumor breast clinical samples, obtaining a map of propionylations and butyrylations found in different tissue contexts. A quantitative histone PTM analysis also revealed a contribution of histone acylations in discriminating different tissues, also upon perturbation with antibiotics, and breast cancer samples from the normal counterpart. Our results show that profiling only classical modifications is limiting and highlight the importance of using sample preparation methods that allow the analysis of the widest possible spectrum of histone modifications, paving the way for deeper insights into their functional significance in cellular processes and disease states.

Prevalence and Molecular Detection of Virulence Genes among Multidrug-Resistant Escherichia coli from Human Clinical Samples and Poultry in Duhok City, Iraq

Abstract Background: Extended-spectrum beta-lactamase (ESBL)-producing, carbapenem-resistant Escherichia coli has increased virulence and multidrug resistance (MDR). Objectives: This study was designed to ascertain the frequency of some virulence factor genes, antibiotic susceptibility patterns, ESBLs, and MDR, focused on colistin-resistant E. coli strains of human and animal origin in Duhok city, Iraq. Materials and Methods: Between December 2020 and April 2021, a total of 150 E. coli isolates (110 from human clinical specimens and 40 from poultry cloacal swabs) were included in this study. The isolates underwent screening for antibiotic susceptibility, MDR, ESBL, and molecular detection of four virulence genes (fimA, cnfL, crL, and hlyA) was conducted using the polymerase chain reaction. Results: Urine specimens (77.2%) compared to blood, wound, vaginal swab, sputum, and semen from outpatients (71.8%). All strains from humans and poultry showed high resistance to ampicillin (86%–97%), ceftriaxone (74%–47%), tetracycline (72%–85%), ciprofloxacin (48%–97%), and colistin (17%–12%). The lowest levels of resistance were found for carbapenems (4%–4%), and the MDR for the isolates was 63%–93%. Apart from carbapenems, colistin-resistant isolates, especially those from poultry, exhibited significant resistance to other antibiotics, and 57% of these isolates being ESBL producers. Three virulence genes (fimA, cnfL, and crL) were highly prevalent (92%) in human isolates, with the crL gene being predominant (100%). Among poultry isolates, fimA was more prevalent (94%) while crL was less common (6%). Conclusion: The predominance of isolates of colistin-resistant poultry origin and the virulence of isolates of human E. coli origin indicate that both strains are currently experiencing an increase in antibiotic resistance.

Phenotypic and genotypic characterization of methicillin and vancomycin-resistant Staphylococcus aureus isolated from human clinical samples in Kerman hospitals, Iran

Today, methicillin-resistant S. aureus (MRSA) isolates are widely spread in both communities and hospitals, causing high morbidity and mortality worldwide. However, the emergence of vancomycin resistance among MRSA isolates has been perceived as a formidable threat to therapeutic management. Therefore, the present study aimed to determine the frequency and antibiotic susceptibility of MRSA and vancomycin-resistant S. aureus (VRSA) strains in Kerman hospitals in Iran. A total of 455 human clinical samples were collected from Kerman hospitals. The samples were examined for the presence of S. aureus and the isolates were tested for their susceptibility to antibiotics by disk diffusion method. MRSA and VRSA isolates were detected by a combination of phenotypic and genotypic methods. Sixty-five coagulase-positive S. aureus strains were isolated, and the higher isolation rate was from wound samples (47.7%) compared with blood (18.5%), respiratory tract secretions (10.8%), urine (10.8%), body fluids (9.2%), abscess (1.5%), and cerebrospinal fluids (1.5%). The isolates exhibited high resistance toward tetracycline (69.2%). Thirty-four isolates (52.3%) were categorized as MRSA (phenotypically resistant to cefoxitin). mecA gene was amplified in 97.1% of MRSA strains. Moreover, only one MRSA strain was resistant to vancomycin (MIC=128), and this isolate carried the vanA gene. The frequency of MRSA was not significantly associated with gender, age, sample type, and sampling locality (p>0.05). Given the alarming rate of resistance among MRSA isolates, monitoring of antibiotic resistance should be performed to reduce treatment failure in patients with staphylococcal infections. Although vancomycin remains a drug of choice for MRSA, our study suggests that its efficacy may be limited by resistance development.

Antimalarial resistance risk in Mozambique detected by a novel quadruplex droplet digital PCR assay.

While the Plasmodium falciparum malaria parasite continues to cause severe disease globally, Mozambique is disproportionally represented in malaria case totals. Acquisition of copy number variations (CNVs) in the parasite genome contributes to antimalarial drug resistance through overexpression of drug targets. Of interest, piperaquine resistance is associated with plasmepsin 2 and 3 CNVs (pfpmp2 and pfpmp3, respectively), while CNVs in the multidrug efflux pump, multidrug resistance-1 (pfmdr1), increase resistance to amodiaquine and lumefantrine. These antimalarials are partner drugs in artemisinin combination therapies (ACTs) and therefore, CNV detection with accurate and efficient tools is necessary to track ACT resistance risk. Here, we evaluated ~300 clinically derived samples collected from three sites in Mozambique for resistance-associated CNVs. We developed a novel, medium-throughput, quadruplex droplet digital PCR (ddPCR) assay to simultaneously quantify the copy number of pfpmp3, pfpmp2, and pfmdr1 loci in these clinical samples. By using DNA from laboratory parasite lines, we show that this nanodroplet-based method is capable of detecting picogram levels of parasite DNA, which facilitates its application for low yield and human host-contaminated clinical surveillance samples. Following ddPCR and the application of quality control standards, we detected CNVs in 13 of 229 high-quality samples (prevalence of 5.7%). Overall, our study revealed a low number of resistance CNVs present in the parasite population across all three collection sites, including various combinations of pfmdr1, pfpmp2, and pfpmp3 CNVs. The potential for future ACT resistance across Mozambique emphasizes the need for continued molecular surveillance across the region.