Unraveling the impact of cancer-associated fibroblasts on hypovascular pancreatic neuroendocrine tumors.

BACKGROUND: Pancreatic neuroendocrine tumors (PNETs) with low microvessel density and fibrosis often exhibit clinical aggressiveness. Given the contribution of cancer-associated fibroblasts (CAFs) to the hypovascular fibrotic stroma in pancreatic ductal adenocarcinoma, investigating whether CAFs play a similar role in PNETs becomes imperative. In this study, we investigated the involvement of CAFs in PNETs and their effects on clinical outcomes. METHODS: We examined 79 clinical PNET specimens to evaluate the number and spatial distribution of α-smooth muscle actin (SMA)-positive cells, which are indicative of CAFs. Then, the findings were correlated with clinical outcomes. In vitro and in vivo experiments were conducted to assess the effects of CAFs (isolated from clinical specimens) on PNET metastasis and growth. Additionally, the role of the stromal-cell-derived factor 1 (SDF1)-AGR2 axis in mediating communication between CAFs and PNET cells was investigated. RESULTS: αSMA-positive and platelet-derived growth factor-α-positive CAFs were detected in the hypovascular stroma of PNET specimens. A higher abundance of α-SMA-positive CAFs within the PNET stroma was significantly associated with a higher level of clinical aggressiveness. Notably, conditioned medium from PNET cells induced an inflammatory phenotype in isolated CAFs. These CAFs promoted PNET growth and metastasis. Mechanistically, PNET cells secreted interleukin-1, which induced the secretion of SDF1 from CAFs. This cascade subsequently elevated AGR2 expression in PNETs, thereby promoting tumor growth and metastasis. The downregulation of AGR2 in PNET cells effectively suppressed the CAF-mediated promotion of PNET growth and metastasis. CONCLUSION: CAFs drive the growth and metastasis of aggressive PNETs. The CXCR4-SDF1 axis may be a target for antistromal therapy in the treatment of PNET. This study clarifies mechanisms underlying PNET aggressiveness and may guide future therapeutic interventions targeting the tumor microenvironment.

Bicyclic Amidine-Triggered Cyclization of o-Alkynylisocyanobenzenes: Synthesis of Lactam-Derived Quinolines.

Efficient access to the synthesis of lactam-derived quinoline through a bicyclic amidine-triggered cyclization reaction from readily prepared o-alkynylisocyanobenzenes has been developed. The reaction was initiated by nucleophilic attack of the bicyclic amidines to o-alkynylisocyanobenzenes, subsequently with intramolecular cyclization to produce a DBU-quinoline-based amidinium salt, followed by hydrolysis to afford the lactam-derived quinoline in moderate to good yields.

Resolution of an insidious and migratory Mycobacterium tuberculosis-associated secondary organizing pneumonia: a case report and literature review.

BACKGROUND: Organizing pneumonia (OP) is a rare interstitial lung disease. Secondary organizing pneumonia (SOP) caused by Mycobacterium tuberculosis (MTB) is extremely rare. Migratory MTB-associated SOP is more deceptive and dangerous. When insidious tuberculosis (TB) is not recognized, SOP would be misdiagnosed as cryptogenic organizing pneumonia (COP). Use of steroid hormone alone leads to the progression of TB foci or even death. Clues of distinguishing atypical TB at the background of OP is urgently needed. CASE PRESENTATION: A 56-year-old female patient was hospitalized into the local hospital because of cough and expectoration for more than half a month. Her medical history and family history showed no relation to TB or other lung diseases. Community-acquired pneumonia was diagnosed and anti-infection therapy was initialized but invalid. The patient suffered from continuous weigh loss. More puzzling, the lesions were migratory based on the chest computed tomography (CT) images. The patient was then transferred to our hospital. The immunological indexes of infection in blood and pathogenic tests in sputum and the bronchoalveolar lavage fluid were negative. The percutaneous lung puncture biopsy and pathological observation confirmed OP, but without granulomatous lesions. Additionally, pathogen detection of the punctured lung tissues by metagenomics next generation sequencing test (mNGS) were all negative. COP was highly suspected. Fortunately, the targeted next-generation sequencing (tNGS) detected MTB in the punctured lung tissues and MTB-associated SOP was definitely diagnosed. The combined therapy of anti-TB and prednisone was administrated. After treatment for 10 days, the partial lesions were significantly resorbed and the patient was discharged. In the follow-up of half a year, the patient was healthy. CONCLUSIONS: It is difficult to distinguish SOP from COP in clinical practice. Diagnosis of COP must be very cautious. Transient small nodules and cavities in the early lung image are a clue to consider TB, even though all pathogen tests are negative. tNGS is also a powerful tool to detect pathogen, ensuring prompt diagnosis of TB-related SOP. For clinicians in TB high burden countries, we encourage them to keep TB in mind before making a final diagnosis of COP.

The first report of co-existence of pulmonary tuberculosis and lung malignancy in a kidney transplant recipient: a case report and literature review.

BACKGROUND: Along with the medical development, organ transplant patients increase dramatically. Since these transplant patients take immunosuppressants for a long term, their immune functions are in a suppressed state, prone to all kinds of opportunistic infections and cancer. However, it is rarely reported that the kidney transplant recipients (KTRs) have pulmonary tuberculosis and lung cancer simultaneously. CASE PRESENTATION: A 60-year-old male was admitted because of persistent lung shadow for 2 years without any obvious symptom 8 years after renal transplant. T-SPOT test was positive but other etiological examinations for Mycobacterium tuberculosis were negative. Chest CT scan revealed two pulmonary lesions in the right upper and lower lobe respectively. 18F-fluorodesoxyglucose positron-emission tomography (FDG-PET) CT found FDG intake increased in both pulmonary consolidation lesions. CT-guided percutaneous transthoracic needle biopsy revealed lung adenocarcinoma and tuberculosis. The video-assisted thoracoscopic surgery was operated to resect the malignancy lesions. The patient received specific anti-tuberculosis therapy and was discharged. At the follow-up of 6 months post drug withdrawal, the patient was recovered very well. CONCLUSIONS: We for the first time reported co-existence of smear-negative pulmonary TB and lung adenocarcinoma in a KTR, which highlighted the clinical awareness of co-occurrence of TB and malignancy after renal transplant and emphasized the value of biopsy and 18F-FDG-PET in early diagnosis of TB and cancer.

Novel cinnamic acid magnolol derivatives as potent α-glucosidase and α-amylase inhibitors: Synthesis, in vitro and in silico studies.

In this study, twenty novel cinnamic acid magnolol derivatives were synthesized, and screened for their anti-hyperglycemic potential. All synthesized compounds exhibited good to moderate α-glucosidase and α-amylase inhibitory activities with IC50 values: 5.11 ± 1.46-90.26 ± 1.85 µM and 4.27 ± 1.51-49.28 ± 2.54 µM as compared to the standard acarbose (IC50: 255.44 ± 1.89 µM and 80.33 ± 2.95 µM, respectively). Compound 6j showed the strongest inhibitory activity against α-glucosidase (IC50 = 5.11 ± 1.46 µM) and α-amylase (IC50 = 4.27 ± 1.51 µM). Kinetic study indicated that compound 6j was reversible and a mixed type inhibitor against α-glucosidase and α-amylase. In silico studies revealed the binding interaction between 6j and two enzymes, respectively. Finally, cells cytotoxicity assay revealed that compound 6j showed low toxicity against 3 T3-L1 cells and HepG2 cells.

Thymidylate kinase is critical for DNA repair via ATM-dependent Tip60 complex formation.

Cellular supply of deoxythymidine triphosphate (dTTP) is crucial for DNA replication and repair. Thymidylate kinase (TMPK) catalyzes the conversion of thymidine monophosphate to thymidine diphosphate, which is an essential step for dTTP synthesis. Despite their major cellular localization in cytosol, TMPK and ribonucleotide reductase (RNR) are detected at DNA damage sites for local dNDP formation. Because deoxyuridine diphosphate is synthesized by RNR, the simultaneous recruitment of TMPK and RNR to DNA damage sites is critical for preventing deoxyuridine triphosphate-mediated toxic repair. This study investigates the mechanism responsible for the recruitment of TMPK to DNA damage sites. Our data demonstrate the requirement of ataxia telangiectasia mutated (ATM) kinase activity for TMPK recruitment to DNA lesion sites. Moreover, we find that TMPK is able to form the complex with histone acetyltransferase Tip60 and RNR. Inhibition of ATM kinase reduces the complex formation and TMPK phosphorylation. Our analysis further shows the presence of TMPK phosphorylation at serine 88, which is an ATM kinase consensus site. A phosphorylation-defective mutation at this site suppresses TMPK recruitment to DNA damage sites and the complex formation with Tip60. Finally, we provide evidence that this site is critical for the function of TMPK in DNA repair but not for catalytic activity. Together, these findings suggest that Tip60-ATM signaling has a functional contribution to the recruitment of TMPK to DNA damage sites, thereby increasing local dTTP synthesis for DNA repair.-Hu, C.-M., Tsao, N., Wang, Y.-T., Chen, Y.-J., Chang, Z.-F. Thymidylate kinase is critical for DNA repair via ATM-dependent Tip60 complex formation.

Correction to: Effect of RAB31 silencing on osteosarcoma cell proliferation and migration through the Hedgehog signaling pathway.

In the original publication of the article, some of the pictures were misplaced in the Fig. 6 and published incorrectly. The correct Fig. 6 is provided in this version.

Deleterious cardiovascular effect of exosome in digitalis-treated decompensated congestive heart failure.

Heart failure (HF) is a medical condition inability of the heart to pump sufficient blood to meet the metabolic demand of the body to take place. The number of hospitalized patients with cardiovascular diseases is estimated to be more than 1 million each year, of which 80% to 90% of patients ultimately progress to decompensated HF. Digitalis glycosides exert modest inotropic actions when administered to patients with decompensated HF. Although its efficacy in patients with HF and atrial fibrillation is clear, its value in patients with HF and sinus rhythm has often been questioned. A series of recent studies have cast serious doubt on the benefit of digoxin when added to contemporary HF treatment. We are hypothesizing the role and mechanism of exosome and its biological constituents responsible for worsening the disease state and mortality in decompensated HF patients on digitalis.

Thrombospondin-2 is a Highly Specific Diagnostic Marker and is Associated with Prognosis in Pancreatic Cancer.

BACKGROUND: Thrombospondin-2 (TSP-2) has been reported as an early diagnostic marker for pancreatic ductal adenocarcinoma (PDAC) in Caucasian populations. This study was designed to validateTSP-2 as a diagnostic marker in a large Taiwan cohort and to investigate the association of TSP-2 with the clinical outcomes of PDAC patients. METHODS: The serum TSP-2 levels in 263 PDAC patients and 230 high-risk individuals (HRIs) were measured via an enzyme-linked immunosorbent assay. The sensitivity, specificity, and accuracy of TSP-2 as a diagnostic marker to discriminating PDAC patients from HRIs and correlations between TSP-2 levels and prognosis of PDAC patients were analyzed. RESULTS: Serum TSP-2 levels were significantly higher in patients with PDAC (44.90 ± 40.70 ng/ml) than in the HRIs (17.52 ± 6.23 ng/ml). At a level of ≥ 29.8 ng/ml, TSP-2 exhibited 100% specificity, 55.9% sensitivity, 100% positive predictive value (PPV), and 66.5% negative predictive value (NPV) for discriminating PDAC patients from HRIs. The Cox regression analysis showed that higher serum TSP-2 levels were significantly associated with poor outcomes in PDAC patients (hazard ratio = 1.54, 95% confidence interval = 1.143-2.086, P = 0.005). Combining the carbohydrate antigen 19-9 (CA19-9) (cutoff value of 62.0 U/ml) and TSP-2 (cutoff value of 29.8 ng/ml) levels yielded 98.7% specificity, 90.5% sensitivity, 98.8% PPV, and 90.1% NPV for discriminating patients with PDAC from HRIs. CONCLUSIONS: TSP-2 is a highly specific diagnostic marker and an independent prognostic marker in patients with PDAC. A combined biomarker panel, including TSP-2 and CA19-9, may facilitate future PDAC screening.

Effect of RAB31 silencing on osteosarcoma cell proliferation and migration through the Hedgehog signaling pathway.

Osteosarcoma (OS) is a prevalent cancer that plagues people worldwide. Identifying prognostic markers would be useful in treating human OS. In this study, we aimed to explore the functions of Ras-related protein Rab-31 (RAB31) in OS-cell proliferation, migration, and invasion as well as its roles in the Hedgehog signaling pathway for better understanding of the mechanism. To assess the detailed regulatory mechanism of RAB31 silencing on OS, both RT-qPCR and Western blot analysis were employed to evaluate the expressions of RAB31 as well as the Hedgehog signaling pathway-related genes. Besides, we also investigated the effects of silenced RAB31 both in vitro and in vivo. First, we found that in OS tissues, both mRNA and protein expressions of RAB31 and PCNA had a significant increase. Second, the Hedgehog signaling pathway was detected to play an integral role in OS progression. Finally, after transfection of RAB31-siRNA to reduce the expression of RAB31, the Hedgehog signaling pathway was suppressed, along with cell proliferation, invasion, and migration. Therefore, we conclude that RAB31 plays an important role in OS development and its silencing delays the OS progression via suppression of the Hedgehog signaling pathway.

Efficacy of different drugs in the treatment of minimal hepatic encephalopathy: A network meta-analysis involving 826 patients based on 10 randomized controlled trials.

Minimal hepatic encephalopathy (MHE), a complex neuropsychological complication of cirrhosis, is characterized by delayed reaction time and the inhibition of abnormal response. This network meta-analysis (NMA) was adopted to compare the efficacy of five drugs including lactulose, probiotics, rifaximin, acetyl-L-carnitine (ALC), and L-Ornithine L-aspartate (LOLA) in the treatment of MHE. The Cochrane Library, PubMed, and Embase databases were searched for any existing entail on these five drugs from the inception to February 2018, including Randomized controlled trials (RCTs). The NMA of the five drugs, accounting for both direct and indirect comparisons to assess WMD (weighted mean difference) and SUCRA (surface under the cumulative ranking curves) was conducted. In total, 10 RTCS with 826 MHE patients met the inclusion criteria and were included in the NMA. The meta-analysis revealed that compared with placebo, lactulose, and probiotics had better efficacy in reducing ammonia serum levels and total SIP (Sickness Impact Profil) score; ALC had better efficacy in lowering serum level of ammonia and increasing the serum level of albumin; rifaximin and LOLA had better efficacy in reducing total SIP score. The results from SUCRA revealed that in terms of ammonia and albumin serums, ALC presented with the highest rankings when it comes to efficacy (serum ammonia: 87.2%; serum albumin: 92.25%). Hence, the key findings from the present study highly suggested that ALC had the best efficacy in the treatment of MHE patients.

Epigenetic silencing of sonic hedgehog elicits antitumor immune response and suppresses tumor growth by inhibiting the hedgehog signaling pathway in metastatic spine tumors in Sprague-Dawley rats.

BACKGROUND: Patients with metastatic spine tumors may suffer from pain or neurologic deficit, and the disease may be detected in patients with a known malignancy. Sonic hedgehog (SHH) has received special attention due to its role in cancers. Therefore, this study investigated the effects of epigenetic silencing of SHH on antitumor immune response and tumor growth by regulating the hedgehog (Hh) signaling pathway in metastatic spine tumors. METHODS: Rat models of metastatic spine tumors were successfully established. We first calculated the tumor volume and the inhibition rate of tumor growth to investigate the effect of SHH on tumor growth. Afterwards, immunohistochemistry was used to determine whether proliferation was delayed by SHH depletion, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was conducted to test the changes in the lymphocyte transformation rate in the spleen triggered by SHH silencing. Then, the influence of SHH depletion on immune function was investigated. Later, quantitative reverse transcription polymerase chain reaction and Western blot assay were performed to explore the Hh signaling pathway-related factors. Finally, we added the Hh signaling pathway inhibitor, GDC-0449, to confirm the role of the pathway in tumor progression. RESULTS: Initially, we observed that SHH depletion was a negative factor for tumor growth. Afterwards, it was revealed that epigenetic silencing of SHH served as an inhibitor factor for the function of spleen lymphocyte transformation and inflammation while promoting antitumor immune function. CONCLUSION: Our preliminary results indicate that epigenetic silencing of SHH elicits an antitumor immune response and suppresses tumor growth by inhibiting the Hh signaling pathway in metastatic spine tumors.

Complementary interhelical interactions between three buried Glu-Lys pairs within three heptad repeats are essential for Hec1-Nuf2 heterodimerization and mitotic progression.

Hec1 and Nuf2, core components of the NDC80 complex, are essential for kinetochore-microtubule attachment and chromosome segregation. It has been shown that both Hec1 and Nuf2 utilize their coiled-coil domains to form a functional dimer; however, details of the consequential significance and structural requirements to form the dimerization interface have yet to be elucidated. Here, we showed that Hec1 required three contiguous heptad repeats from Leu-324 to Leu-352, but not the entire first coiled-coil domain, to ensure overall stability of the NDC80 complex through direct interaction with Nuf2. Substituting the hydrophobic core residues, Leu-331, Val-338, and Ile-345, of Hec1 with alanine completely eliminated Nuf2 binding and blocked mitotic progression. Moreover, unlike most coiled-coil proteins, where the buried positions are composed of hydrophobic residues, Hec1 possessed an unusual distribution of glutamic acid residues, Glu-334, Glu-341, and Glu-348, buried within the interior dimerization interface, which complement with three Nuf2 lysine residues: Lys-227, Lys-234, and Lys-241. Substituting these corresponding residues with alanine diminished the binding affinity between Hec1 and Nuf2, compromised NDC80 complex formation, and adversely affected mitotic progression. Taken together, these findings demonstrated that three buried glutamic acid-lysine pairs, in concert with hydrophobic interactions of core residues, provide the major specificity and stability requirements for Hec1-Nuf2 dimerization and NDC80 complex formation.

[Wuling capsule played an assistant role in primary prevention of post-stroke depression: a clinical research].

OBJECTIVE: To observe the primary prevention role of Wuling Capsule (WC) on poststroke depression (PSD) patients. METHODS: Acute stroke patients were recruited and randomized into 2 groups by stratification, 55 in each group. All patients received same routine treatment of cardiovascular diseases. Patients in the experimental group additionally took WC (0.33 g each pill), 3 pills per day, three times per day; while those in the control group additionally took placebos, 3 pills per day, three times per day. Two weeks consisted of one therapeutic course. The diagnosis of PSD was performed once every other week. Those in accordance with PSD diagnosis discontinued any drug therapy. Those not in accordance with PSD diagnosis continued the drug therapy for 1-12 therapeutic course(s) (in total of 6 months). If they were still not in accordance with PSD diagnosis, then they discontinued the drug therapy. The morbidity of PSD, the average time of depression occurrence, Hamilton depression rating scale (HAMD) score, and adverse reactions were observed. RESULTS: The 1-, 3-, and 6-month morbidity of PSD was 8%, 16%, and 34% in the experimental group, while they were 19.6%, 29.4%, and 54.9% in the control group. The occurrence rate was lower in the experimental group than in the control group. Besides, there was statistical difference in the 6-month occurrence rate between the two groups (chi2 = 4.465, P < 0.05). The average time of PSD occurrence was longer in the experimental group than in the control group (14.96 +/- 8.31 weeks vs. 9.36 +/- 6.06 weeks; t=6.762, P < 0.05). The HAMD score at the PSD occurrence was 11.96 +/- 2.14 in the experimental group, lower than that of the control group (14.57 +/- 4.24), showing statistical difference (t=5.641, P < 0.05). CONCLUSION: WC was superior to the placebos in lowering the incidence of PSD, delaying the occurrence time of PSD, attenuating the depression degree of PSD, and had certain preventive effect on the incidence of PSD.

Dual-Mechanism Peptide SR25 has Broad Antimicrobial Activity and Potential Application for Healing Bacteria-infected Diabetic Wounds.

The rise of antibiotic resistance poses a significant public health crisis, particularly due to limited antimicrobial options for the treatment of infections with Gram-negative pathogens. Here, an antimicrobial peptide (AMP) SR25 is characterized, which effectively kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism without detectable resistance. Meanwhile, an SR25-functionalized hydrogel is developed for the efficient treatment of infected diabetic wounds. SR25 is obtained through genome mining from an uncultured bovine enteric actinomycete named Nonomuraea Jilinensis sp. nov. Investigations reveal that SR25 has two independent cellular targets, disrupting bacterial membrane integrity and restraining the activity of succinate:quinone oxidoreductase (SQR). In a diabetic mice wound infection model, the SR25-incorporated hydrogel exhibits high efficacy against mixed infections of Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA), accelerating wound healing. Overall, these findings demonstrate the therapeutic potential of SR25 and highlight the value of mining drugs with multiple mechanisms from uncultured animal commensals for combating challenging bacterial pathogens.

Comprehending the inhibition mechanism of indole-based bis-acylhydrazone compounds on α-glucosidase: Spectral and theoretical approaches.

Indole-based bis-acylhydrazone compounds can inhibit the activity of α-glucosidase and control the concentration of blood glucose. In this paper, the characteristics of three indole-based bis-acylhydrazone compounds with different inhibitory activities of α-glucosidase as well as the interaction with α-glucosidase were studied by experiments and computational simulation techniques. Enzyme kinetic and spectral experiments showed that the indole-based bis-acylhydrazone compounds were able to inhibit enzyme activity through mixed inhibition dominated by competitive inhibition, and during the binding reaction, indole-based bis-acylhydrazone compounds can quench the intrinsic fluorescence of α-glucosidase through static quenching and an aggregation of the indole-based bis-acylhydrazone with α-glucosidase produces a stable complex with a molar ratio of 1:1, and the combination of indole-based bis-acylhydrazone compounds could lead to slight change in the conformation of α-glucosidase. The theoretical simulation demonstrated that the stability of the complex systems was positively correlated with the inhibitory activity of indole-based bis-acylhydrazone compounds, and the indole-based bis-acylhydrazone compounds occupied the active site in the multi-ligand system, resulting in a significant decrease in the binding ability of starch to active amino acids. These results suggested that indole-based bis-acylhydrazone compound was expected to be a new type of α-glucosidase inhibitor.

The potential association between metabolic disorders and pulmonary tuberculosis: a Mendelian randomization study.

BACKGROUND: Metabolic disorders (MetDs) have been demonstrated to be closely linked to numerous diseases. However, the precise association between MetDs and pulmonary tuberculosis (PTB) remains poorly understood. METHOD: Summary statistics for exposure and outcomes from genome-wide association studies (GWASs) for exposures and outcomes were obtained from the BioBank Japan Project (BBJ) Gene-exposure dataset. The 14 clinical factors were categorized into three groups: metabolic laboratory markers, blood pressure, and the MetS diagnostic factors. The causal relationship between metabolic factors and PTB were analyzed using two-sample Mendelian Randomization (MR). Additionally, the direct effects on the risk of PTB were investigated through multivariable MR. The primary method employed was the inverse variance-weighted (IVW) model. The sensitivity of this MR analysis was evaluated using MR-Egger regression and the MR-PRESSO global test. RESULTS: According to the two-sample MR, HDL-C, HbA1c, TP, and DM were positively correlated with the incidence of active TB. According to the multivariable MR, HDL-C (IVW: OR 2.798, 95% CI 1.484-5.274, P = 0.001), LDL (IVW: OR 4.027, 95% CI 1.140-14.219, P = 0.03) and TG (IVW: OR 2.548, 95% CI 1.269-5.115, P = 0.009) were positively correlated with the occurrence of PTB. TC (OR 0.131, 95% CI 0.028-0.607, P = 0.009) was negatively correlated with the occurrence of PTB. We selected BMI, DM, HDL-C, SBP, and TG as the diagnostic factors for metabolic syndrome. DM (IVW, OR 1.219, 95% CI 1.040-1.429 P = 0.014) and HDL-C (IVW, OR 1.380, 95% CI 1.035-1.841, P = 0.028) were directly correlated with the occurrence of PTB. CONCLUSIONS: This MR study demonstrated that metabolic disorders, mainly hyperglycemia, and dyslipidemia, are associated with the incidence of active pulmonary tuberculosis.

ApoE Mimetic Peptide COG1410 Kills Mycobacterium smegmatis via Directly Interfering ClpC's ATPase Activity.

Antimicrobial peptides (AMPs) hold promise as alternatives to combat bacterial infections, addressing the urgent global threat of antibiotic resistance. COG1410, a synthetic peptide derived from apolipoprotein E, has exhibited potent antimicrobial properties against various bacterial strains, including Mycobacterium smegmatis. However, our study reveals a previously unknown resistance mechanism developed by M. smegmatis against COG1410 involving ClpC. Upon subjecting M. smegmatis to serial passages in the presence of sub-MIC COG1410, resistance emerged. The comparative genomic analysis identified a point mutation in ClpC (S437P), situated within its middle domain, which led to high resistance to COG1410 without compromising bacterial fitness. Complementation of ClpC in mutant restored bacterial sensitivity. In-depth analyses, including transcriptomic profiling and in vitro assays, uncovered that COG1410 interferes with ClpC at both transcriptional and functional levels. COG1410 not only stimulated the ATPase activity of ClpC but also enhanced the proteolytic activity of Clp protease. SPR analysis confirmed that COG1410 directly binds with ClpC. Surprisingly, the identified S437P mutation did not impact their binding affinity. This study sheds light on a unique resistance mechanism against AMPs in mycobacteria, highlighting the pivotal role of ClpC in this process. Unraveling the interplay between COG1410 and ClpC enriches our understanding of AMP-bacterial interactions, offering potential insights for developing innovative strategies to combat antibiotic resistance.

Engineered endolysin of Klebsiella pneumoniae phage is a potent and broad-spectrum bactericidal agent against "ESKAPEE" pathogens.

The rise of antimicrobial resistance in ESKAPEE pathogens poses significant clinical challenges, especially in polymicrobial infections. Bacteriophage-derived endolysins offer promise in combating this crisis, but face practical hurdles. Our study focuses on engineering endolysins from a Klebsiella pneumoniae phage, fusing them with ApoE23 and COG133 peptides. We assessed the resulting chimeric proteins' bactericidal activity against ESKAPEE pathogens in vitro. ApoE23-Kp84B (CHU-1) reduced over 3 log units of CFU for A. baumannii, E. faecalis, K. pneumoniae within 1 h, while COG133-Kp84B (CHU-2) showed significant efficacy against S. aureus. COG133-L1-Kp84B, with a GS linker insertion in CHU-2, exhibited outstanding bactericidal activity against E. cloacae and P. aeruginosa. Scanning electron microscopy revealed alterations in bacterial morphology after treatment with engineered endolysins. Notably, CHU-1 demonstrated promising anti-biofilm and anti-persister cell activity against A. baumannii and E. faecalis but had limited efficacy in a bacteremia mouse model of their coinfection. Our findings advance the field of endolysin engineering, facilitating the customization of these proteins to target specific bacterial pathogens. This approach holds promise for the development of personalized therapies tailored to combat ESKAPEE infections effectively.