A novel SIK2 inhibitor SIC-19 exhibits synthetic lethality with PARP inhibitors in ovarian cancer.

PURPOSE: Ovarian cancer patients with HR proficiency (HRP) have had limited benefits from PARP inhibitor treatment, highlighting the need for improved therapeutic strategies. In this study, we developed a novel SIK2 inhibitor, SIC-19, and investigated its potential to enhance the sensitivity and expand the clinical utility of PARP inhibitors in ovarian cancer. METHODS: The SIK2 protein was modeled using a Molecular Operating Environment (MOE), and the most favorable model was selected based on a GBVI/WSA dG scoring function. The Chembridge Compound Library was screened, and the top 20 candidate compounds were tested for their interaction with SIK2 and downstream substrates, AKT-pS473 and MYLK-pS343. SIC-19 emerged as the most promising drug candidate and was further evaluated using multiple assays. RESULTS: SIC-19 exhibited selective and potent inhibition of SIK2, leading to its degradation through the ubiquitination pathway. The IC50 of SIC-19 correlated inversely with endogenous SIK2 expression in ovarian cancer cell lines. Treatment with SIC-19 significantly inhibited cancer cell growth and sensitized cells to PARP inhibitors in vitro, as well as in ovarian cancer organoids and xenograft models. Mechanistically, SIK2 knockdown and SIC-19 treatment reduced RAD50 phosphorylation at Ser635, prevented nuclear translocation of RAD50, disrupted nuclear filament assembly, and impaired DNA homologous recombination repair, ultimately inducing apoptosis. These findings highlight the crucial role of SIK2 in the DNA HR repair pathway and demonstrate the significant PARP inhibitor sensitization achieved by SIC-19 in ovarian cancer. CONCLUSIONS: SIC-19, a novel SIK2 inhibitor, effectively inhibits tumor cell growth in ovarian cancer by interfering with RAD50-mediated DNA HR repair. Furthermore, SIC-19 enhances the efficacy of PARP inhibitors, providing a promising therapeutic strategy to improve outcomes for ovarian cancer patients.

Oral and oropharyngeal NUT carcinoma: a multicentre screening study of poorly differentiated oral cancer.

BACKGROUND AND AIMS: Nuclear protein testis (NUT) carcinoma (NC) is a rare and highly aggressive tumour characterised by chromosomal rearrangement of the nuclear protein testis family member 1 (NUTM1) gene, also known as the NUT gene. NC occurs mainly in the head and neck, mediastinum and lung. In general, primary NC in the oral cavity is extremely rare and reported sporadically. METHODS: A total of 111 formalin-fixed and paraffin-embedded specimens of poorly differentiated oral and oropharyngeal tumours were collected from 10 hospitals. NUT protein IHC staining was performed on these samples, and fluorescence in-situ hybridisation (FISH) and RNA sequencing detection were further carried out for NUT IHC-positive cases. RESULTS: The expression of NUT protein in tumour cells was detected in five cases (five of 111, 4.5%). The tumours in these cases were located in the oral floor, lip, base of the tongue, gingiva and hard palate. FISH detection results showed BRD4::NUT rearrangement in three patients and a non-BRD4::NUT rearrangement pattern in two patients. RNA sequencing results confirmed BRD4::NUT rearrangement in two cases. CONCLUSIONS: To our knowledge, this is the first and largest retrospective study of oral NC, and we found that NC is easily misdiagnosed as poorly differentiated oral squamous cell carcinoma (SCC) or poorly differentiated carcinoma. The morphology and immunophenotype of four NC cases were similar to SCC, and abrupt keratinisation was observed in three cases. Therefore, it is necessary to detect NUT protein for NC screening in oral malignant tumours with these morphologies, especially for young patients who are more likely to be misdiagnosed with other types of cancer.

Maternally transmitted nonsyndromic hearing impairment may be associated with mitochondrial tRNAAla 5601C>T and tRNALeu(CUN) 12311T>C mutations.

BACKGROUND: Sequence alternations in mitochondrial genomes, especially in genes encoding mitochondrial tRNA (mt-tRNA), were the important contributors to nonsyndromic hearing loss (NSHL); however, the molecular mechanisms remained largely undetermined. METHODS: A maternally transmitted Chinese pedigree with NSHL underwent clinical, genetic, and biochemical assessment. PCR and direct sequence analyses were performed to detect mitochondrial DNA (mtDNA), GJB2, and SLC26A4 gene mutations from matrilineal relatives of this family. Mitochondrial functions including mitochondrial membrane potential (MMP), ATP, and ROS were evaluated in polymononuclear leukocytes (PMNs) derived from three deaf patients and three controls from this pedigree. RESULTS: Four of nine matrilineal relatives developed hearing loss at the variable age of onset. Two putative pathogenic mutations, m.5601C>T in tRNAAla and m.12311T>C in tRNALeu(CUN) , were identified via PCR-Sanger sequencing, as well as 34 variants that belonged to mtDNA haplogroup G2b2. Intriguingly, m.5601C>T mutation resided at very conserved nucleotide in the TψC loop of tRNAAla (position 59), while the T-to-C substitution at position 12311 located at position 48 in the variable stem of tRNALeu(CUN) and was believed to alter the aminoacylation and the steady-state level of tRNA. Biochemical analysis revealed the impairment of mitochondrial functions including the significant reductions of ATP and MMP, whereas markedly increased ROS levels were found in PMNs derived from NSHL patients with m.5601C>T and m.12311T>C mutations. However, we did not detect any mutations in GJB2 and SLC26A4 genes. CONCLUSION: Our data indicated that mt-tRNAAla m.5601C>T and tRNALeu(CUN) 12311T>C mutations were associated with NSHL.

Protein kinase C ϵ stabilizes ß-catenin and regulates its subcellular localization in podocytes.

Kidney disease has been linked to dysregulated signaling via PKC in kidney cells such as podocytes. PKCα is a conventional isoform of PKC and a well-known binding partner of ß-catenin, which promotes its degradation. ß-Catenin is the main effector of the canonical Wnt pathway and is critical in cell adhesion. However, whether other PKC isoforms interact with ß-catenin has not been studied systematically. Here we demonstrate that PKCϵ-deficient mice, which develop proteinuria and glomerulosclerosis, display lower ß-catenin expression compared with PKC wild-type mice, consistent with an altered phenotype of podocytes in culture. Remarkably, ß-catenin showed a reversed subcellular localization pattern: Although ß-catenin exhibited a perinuclear pattern in undifferentiated wild-type cells, it predominantly localized to the nucleus in PKCϵ knockout cells. Phorbol 12-myristate 13-acetate stimulation of both cell types revealed that PKCϵ positively regulates ß-catenin expression and stabilization in a glycogen synthase kinase 3ß-independent manner. Further, ß-catenin overexpression in PKCϵ-deficient podocytes could restore the wild-type phenotype, similar to rescue with a PKCϵ construct. This effect was mediated by up-regulation of P-cadherin and the ß-catenin downstream target fascin1. Zebrafish studies indicated three PKCϵ-specific phosphorylation sites in ß-catenin that are required for full ß-catenin function. Co-immunoprecipitation and pulldown assays confirmed PKCϵ and ß-catenin as binding partners and revealed that ablation of the three PKCϵ phosphorylation sites weakens their interaction. In summary, we identified a novel pathway for regulation of ß-catenin levels and define PKCϵ as an important ß-catenin interaction partner and signaling opponent of other PKC isoforms in podocytes.

Case report: A rare case of coexistence of low-grade appendiceal mucinous neoplasia and goblet cell adenocarcinoma in the appendix.

Background: Primary appendiceal tumors are rare. Low-grade appendiceal mucinous neoplasia (LAMN) and goblet cell adenocarcinoma (GCA) account for 20% and 14% of primary appendiceal tumors, respectively. The coexistence of LAMN and GCA is an extremely rare event. This report presents a case of an elderly male patient with an appendiceal tumor composed of LAMN and GCA in the same appendix. Case presentation: A 72-year-old male patient was admitted to our institution presenting with a history of abdominal pain localized to the right lower quadrant for two months. Abdominal computed tomography (CT) showed a large dilated thickened cystic mass in the appendix, along with a small duodenal diverticulum. Laboratory tests indicated elevated levels of serum carcinoembryonic antigen (CEA) and cancer antigen 199 (CA19-9) markers. The patient underwent a laparoscopic right hemicolectomy and exploration of the duodenal diverticulum, and there was no finding of perforation of the duodenal diverticulum. Focal positivity for chromogranin A (CgA) and synaptophysin (Syn) was observed in the tumor cells of GCA. The final pathological diagnosis revealed the coexistence of LAMN staged pT4a and grade 1 GCA staged pT3 in the appendix. Unfortunately, the patient died due to severe septic shock and circulatory failure secondary to a perforated duodenal diverticulum. Conclusions: The coexistence of LAMN and GCA are extremely rare in the appendix and may result from the proliferation of two independent cellular lines. The coexistence of distinct neoplasms poses diagnostic and management challenges. Multidisciplinary team discussion may be essential in the effective management of these patients.

Joint DNA-RNA-based NGS for diagnosis and treatment of a rare CD47-MET fusion lung adenocarcinoma which was immunoresistant and savoltinib-sensitive: a case report.

Targeted therapy and immunotherapy are both important in the treatment of non-small-cell lung cancer (NSCLC). Accurate diagnose and precise treatment are key in achieving long survival of patients. MET fusion is a rare oncogenic factor, whose optimal detection and treatment are not well established. Here, we report on a 32-year-old female lung adenocarcinoma patient with positive PD-L1 and negative driver gene detected by DNA-based next-generation sequencing (NGS). A radical resection of the primary lesion after chemotherapy combined with PD-1 checkpoint inhibitor administration indicated primary immuno-resistance according to her pathological response and rapid relapse. A rare CD47-MET was detected by RNA-based NGS, which was confirmed by fluorescence in situ hybridization. Multiplex immunofluorescence revealed a PD-L1 related heterogeneous immunosuppressive microenvironment with little distribution of CD4+ T cells and CD8+ T cells. Savolitinib therapy resulted in a progression-free survival (PFS) of >12 months, until a new secondary resistance mutation in MET p.D1228H was detected by re-biopsy and joint DNA-RNA-based NGS after disease progression. In this case, CD47-MET fusion NSCLC was primarily resistant to immunotherapy, sensitive to savolitinib, and developed secondary MET p.D1228H mutation after targeted treatment. DNA-RNA-based NGS is useful in the detection of such molecular events and tracking of secondary mutations in drug resistance. To this end, DNA-RNA-based NGS may be of better value in guiding precise diagnosis and individualized treatment in this patient population.

Functional analysis of the novel mitochondrial tRNATrp and tRNASer(AGY) variants associated with type 2 diabetes mellitus.

BACKGROUND: Mutations in mitochondrial tRNA (mt-tRNA) genes that result in mitochondrial dysfunction play important roles in type 2 diabetes mellitus (T2DM). We pre-viously reported a large Chinese pedigree with maternally inherited T2DM that harbors novel mt-tRNA Trp A5514G and tRNA Ser(AGY) C12237T variants, however, the effects of these mt-tRNA variants on T2DM progression are largely unknown. AIM: To assess the potential pathogenicity of T2DM-associated m.A5514G and m.C12237T variants at genetic, molecular, and biochemical levels. METHODS: Cytoplasmic hybrid (cybrid) cells carrying both m.A5514G and m.C12237T variants, and healthy control cells without these mitochondrial DNA (mtDNA) variants were generated using trans-mitochondrial technology. Mitochondrial features, including mt-tRNA steady-state level, levels of adenosine triphosphate (ATP), mitochondrial membrane potential (MMP), reactive oxygen species (ROS), mtDNA copy number, nicotinamide adenine dinucleotide (NAD+)/NADH ratio, enzymatic activities of respiratory chain complexes (RCCs), 8-hydroxy-deo-xyguanine (8-OhdG), malondialdehyde (MDA), and superoxide dismutase (SOD) were examined in cell lines with and without these mt-tRNA variants. RESULTS: Compared with control cells, the m.A5514G variant caused an approximately 35% reduction in the steady-state level of mt-tRNA Trp (P < 0.0001); however, the m.C12237T variant did not affect the mt-tRNA Ser(AGY) steady-state level (P = 0.5849). Biochemical analysis revealed that cells with both m.A5514G and m.C12237T variants exhibited more severe mitochondrial dysfunctions and elevated oxidative stress than control cells: ATP, MMP, NAD+/NADH ratio, enzyme activities of RCCs and SOD levels were markedly decreased in mutant cells (P < 0.05 for all measures). By contrast, the levels of ROS, 8-OhdG and MDA were significantly increased (P < 0.05 for all measures), but mtDNA copy number was not affected by m.A5514G and m.C12237T variants (P = 0.5942). CONCLUSION: The m.A5514G variant impaired mt-tRNA Trp metabolism, which subsequently caused mitochondrial dysfunction. The m.C12237T variant did not alter the steady-state level of mt-tRNA Ser(AGY), indicating that it may be a modifier of the m.A5514G variant. The m.A5514G variant may exacerbate the pathogenesis and progression of T2DM in this Chinese pedigree.

The Association Between Mitochondrial tRNAGlu Variants and Hearing Loss: A Case-Control Study.

Purpose: This study aimed to examine the frequencies of mt-tRNAGlu variants in 180 pediatric patients with non-syndromic hearing loss (NSHL) and 100 controls. Methods: Sanger sequencing was performed to screen for mt-tRNAGlu variants. These mitochondrial DNA (mtDNA) pathogenic mutations were further assessed using phylogenetic conservation and haplogroup analyses. We also traced the origins of the family history of probands carrying potential pathogenic mtDNA mutations. Mitochondrial functions including mtDNA content, ATP and reactive oxygen species (ROS) were examined in cells derived from patients carrying the mt-tRNAGlu A14692G and CO1/tRNASer(UCN) G7444A variants and controls. Results: We identified four possible pathogenic variants: m.T14709C, m.A14683G, m.A14692G and m.A14693G, which were found in NSHL patients but not in controls. Genetic counseling suggested that one child with the m.A14692G variant had a family history of NSHL. Sequence analysis of mtDNA suggested the presence of the CO1/tRNASer(UCN) G7444A and mt-tRNAGlu A14692G variants. Molecular analysis suggested that, compared with the controls, patients with these variants exhibited much lower mtDNA copy numbers, ATP production, whereas ROS levels increased (p<0.05 for all), suggesting that the m.A14692G and m.G7444A variants led to mitochondrial dysfunction. Conclusion: mt-tRNAGlu variants are important risk factors for NSHL.

The main aim of our study was to explore the association between the mt-tRNA^Glu variants and hearing loss. We found that m.T14709C, m.A14683G, m.A14692G and m.A14693G variants were associated with hearing impairments, these variants localized at extremely conserved nucleotides of mt-tRNA^Glu and may result a failure in tRNA metabolism, furthermore, patients with mt-tRNA^Glu variants exhibited much lower levels of mtDNA copy number, ATP as compared with controls, whereas ROS increased. As a result, mt-tRNA^Glu variants may serve as biomarkers for mitochondrial deafness, and screening for tRNA^Glu variants is recommended for early detection and diagnosis of mitochondrial deafness.

Hidden Infection in Asymptomatic Congenital Lung Malformations-A Decade Retrospective Study.

Background: Whether to operate on asymptomatic patients with congenital lung malformations (CLMs) remains controversial. Our study intended to find out the proportion of hidden infection in CLMs and its effect on surgery, to provide help for the management of asymptomatic CLMs patients. Methods: A retrospective review of the medical records of patients with asymptomatic CLMs from January 2011 to December 2020 was performed in our center. Selected asymptomatic patients were divided into a non-hidden infection group (NHI) and a hidden infection group (HI). Results: A total of 581 asymptomatic CLMs patients were included in this study. Thirty-two percent of asymptomatic CLMs patients had hidden infection in the lesion. Among various CLMs diseases, intralobular pulmonary sequestration had the highest percentage of hidden infection (48.8%). With age, the proportion of HI gradually increased. Patients in the HI and NHI groups were 223 and 121. The incidence of pleural adhesion and focal abscess in the HI group were 14.9 and 7.4%. Statistical significances were shown between the two groups in intraoperative blood loss (p = 0.002), operation time (p = 0.045), chest tube drainage time (p < 0.001), postoperative hospital stay (p < 0.001), and air leak (p = 0.012). Conclusion: The proportion of HI detected by postoperative pathological results was high and they could increase the difficulty and risk of surgery. Therefore, early surgery may be a more appropriate choice for the management of asymptomatic CLMs patients.

Determination of Fentanyl, Alpha-Methylfentanyl, Beta-Hydroxyfentanyl and the Metabolite Norfentanyl in Rat Urine by LC-MS-MS.

Fentanyl and its analogs are potent synthetic opioids with a high potential for abuse and dependence. They have become major contributors to opioid deaths. This study aimed to determine whether the metabolites of fentanyl, alpha-methylfentanyl and beta-hydroxyfentanyl, excreted in the urine, can demonstrate historical drug exposure. Fentanyl is primarily metabolized via CYP3A4 into norfentanyl, although there is little research on its metabolization into alpha-methylfentanyl and beta-hydroxyfentanyl. We conducted in vitro experiments with human liver microsomes (HLMs) and rat liver microsomes (RLMs) to elucidate the major metabolic pathways of alpha-methylfentanyl and beta-hydroxyfentanyl using ultra-high-performance liquid chromatography coupled with mass spectrometry. The results showed that both alpha-methylfentanyl and beta-hydroxyfentanyl were predominantly metabolized into norfentanyl in HLM and RLM. Urine samples were collected at different intervals from 0 h to 72 h after intravenous administration of alpha-methylfentanyl and beta-hydroxyfentanyl (20 µg/kg) to Sprague-Dawley rats. We prepared the samples by liquid-liquid extraction, and the internal standard (IS) was cariprazine. A sensitive, rapid liquid chromatography-tandem mass spectrometry method was developed and validated to determine four analytes in the urine. The lower limit of qualification in urine was 2 pg/mL for fentanyl, 5 pg/mL for alpha-methylfentanyl, 10 pg/mL for beta-hydroxyfentanyl and 40 pg/mL for norfentanyl. The analytical range was 0.002-2 ng/mL for fentanyl, 0.005-5 ng/mL for alpha-methylfentanyl, 0.01-10 ng/mL for beta-hydroxyfentanyl and 0.04-40 ng/mL for norfentanyl. All analytes demonstrated good linearity (R2 > 0.99). The extraction recoveries were in the 67.8%-92.1% range, and the IS-normalized matrix effects were between 55.5% and 74.0% (coefficient of variance < 15%). Our data indicated that norfentanyl has a higher concentration in rat urine and was detectable for at least 3 days after exposure to these compounds. This developed method may be useful in various fields, including forensic analysis, workplace drug testing and monitoring drug abuse.

SIK2 promotes ovarian cancer cell motility and metastasis by phosphorylating MYLK.

Salt-inducible kinase 2 (SIK2; also known as serine/threonine-protein kinase SIK2) is overexpressed in several cancers and has been implicated in cancer progression. However, the mechanisms by which SIK2 regulates cancer cell motility, migration and metastasis in ovarian cancer have not been fully discovered. Here, we identify that SIK2 promotes ovarian cancer cell motility, migration and metastasis in vitro and in vivo. Mechanistically, SIK2 regulated cancer cell motility and migration by myosin light chain kinase, smooth muscle (MYLK)-meditated phosphorylation of myosin light chain 2 (MYL2). SIK2 directly phosphorylated MYLK at Ser343 and activated its downstream effector MYL2, promoting ovarian cancer cell motility and metastasis. In addition, we found that adipocytes induced SIK2 phosphorylation at Ser358 and MYLK phosphorylation at Ser343, enhancing ovarian cancer cell motility. Moreover, SIK2 protein expression was positively correlated with the expression of MYLK-pS343 in ovarian cancer cell lines and tissues. The co-expression of SIK2 and MYLK-pS343 was associated with reduced median overall survival in human ovarian cancer samples. Taken together, SIK2 positively regulates ovarian cancer motility, migration and metastasis, suggesting that SIK2 is a potential candidate for ovarian cancer treatment.

The roles of mitochondrial tRNA mutations in non-dystrophic myotonias.

According a recent report by Heidari et al., a mutational screening for candidate pathogenic mitochondrial tRNA (mt-tRNA) mutations were performed in 45 Iranian patients with non-dystrophic myotonia (NDM) and 70 control subjects. Through PCR amplification and direct sequence analysis, nine mt-tRNA mutations were identified: tRNAMet T4454C, tRNATrp A5568G, tRNACys T5794C, tRNAArg A10438T and T10462C, tRNALeu(CUN) A12308G, tRNAThr A15907G, A15924G and G15928A. However, through the database searches and phylogenetic conservation analysis, we noticed that the tRNAThr A15924G, G15928A and tRNALeu(CUN) A12308G mutations should be classified 'pathogenic'. Thus, the roles of mt-tRNA mutations in clinical expression of NDM needed to be further experimentally addressed.

Was Mafeisan an Anesthetic in Ancient China?

According to the Chinese historical books, Records of the Three Kingdoms () and Book of the Later Han (), Hua Tuo (, 140 - 208), a Traditional Chinese medicine (TCM) physician invented Mafeisan, an oral herbal general anesthetic, more than 1800 years ago during Eastern Han Dynasty. However, no written record of ingredients of the original Mafeisan has been found anywhere so far although there have been several similar anesthetic prescriptions published in TCM books later. There has been controversy over the existence of Mafeisan and even Hua Tuo in Chinese literature. We did extensive literature search and analysis, and believe that there indeed was Mafeisan in Hua Tuo's time.

Role of protein kinase C in podocytes and development of glomerular damage in diabetic nephropathy.

The early glomerular changes in diabetes include a podocyte phenotype with loss of slit diaphragm proteins, changes in the actin cytoskeleton and foot process architecture. This review focuses on the role of the protein kinase C (PKC) family in podocytes and points out the differential roles of classical, novel, and atypical PKCs in podocytes. Some PKC isoforms are indispensable for proper glomerular development and slit diaphragm maintenance, whereas others might be harmful when activated in the diabetic milieu. Therefore, some might be interesting treatment targets in the early phase of diabetes.

Super fast detection of latent fingerprints with water soluble CdTe quantum dots.

A new method based on the use of highly fluorescent water-soluble cadmium telluride (CdTe) quantum dots (QDs) capped with mercaptosuccinic acid (MSA) was explored to develop latent fingerprints. After optimized the effectiveness of QDs method contains pH value and developing time, super fast detection was achieved. Excellent fingerprint images were obtained in 1-3s after immersed the latent fingerprints into quantum dots solution on various non-porous surfaces, i.e. adhesive tape, transparent tape, aluminum foil and stainless steel. High sensitivity of the new latent fingerprints develop method was obtained by developing the fingerprints pressed on aluminum foil successively with the same finger. Compared with methyl violet and rhodamine 6G, the MSA-CdTe QDs showed the higher develop speed and fingerprint image quality. Clear image can be maintained for months by extending exposure time of CCD camera, storing fingerprints in a low temperature condition and secondary development.

Application of mercaptosuccinic acid capped CdTe quantum dots for latent fingermark development.

The aqueous synthesis of mercaptosuccinic acid (MSA) capped CdTe quantum dots (QDs) solution for quickly and sensitively developing latent fingermarks is described. The rapid growth mechanism of CdTe/MSA QDs, which depends on the molecule structure of MSA, is briefly discussed and compared with that of thioglycolic acid (TGA) and mercaptopropionic acid (MPA) capped CdTe QDs. Development of latent fingermarks with the synthesized CdTe/MSA QDs was faster and the ridge details were clearer compared with CdTe/TGA QDs. In addition, latent fingermarks developed with CdTe/MSA QDs showed less background and better contrast than that of gentian violet or rhodamine 6G. Latent fingermarks could be well developed on black tape, scotch tape, tinfoil, aluminum alloy, stainless steel as well as on the adhesive side of yellow tape, even when the latter were aged up to seven days. As immersion time greatly reduced to 10 s by using CdTe/MSA QDs, a preliminary result of latent fingermark development by spraying was presented also.