Hybrid sequence-based analysis reveals the distribution of bacterial species and genes in the oral microbiome at a high resolution.

Bacteria in the oral microbiome are poorly identified owing to the lack of established culture methods for them. Thus, this study aimed to use culture-free analysis techniques, including bacterial single-cell genome sequencing, to identify bacterial species and investigate gene distribution in saliva. Saliva samples from the same individual were classified as inactivated or viable and then analyzed using 16S rRNA sequencing, metagenomic shotgun sequencing, and bacterial single-cell sequencing. The results of 16S rRNA sequencing revealed similar microbiota structures in both samples, with Streptococcus being the predominant genus. Metagenomic shotgun sequencing showed that approximately 80 % of the DNA in the samples was of non-bacterial origin, whereas single-cell sequencing showed an average contamination rate of 10.4 % per genome. Single-cell sequencing also yielded genome sequences for 43 out of 48 wells for the inactivated samples and 45 out of 48 wells for the viable samples. With respect to resistance genes, four out of 88 isolates carried cfxA, which encodes a ß-lactamase, and four isolates carried erythromycin resistance genes. Tetracycline resistance genes were found in nine bacteria. Metagenomic shotgun sequencing provided complete sequences of cfxA, ermF, and ermX, whereas other resistance genes, such as tetQ and tetM, were detected as fragments. In addition, virulence factors from Streptococcus pneumoniae were the most common, with 13 genes detected. Our average nucleotide identity analysis also suggested five single-cell-isolated bacteria as potential novel species. These data would contribute to expanding the oral microbiome data resource.

Current Status and Molecular Mechanisms of Resistance to Immunotherapy in Oral Malignant Melanoma.

Immune checkpoint inhibitors (ICIs), including anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and anti-programmed death-1 (PD-1) antibodies, have initiated a new era in the treatment of malignant melanoma. ICIs can be used in various settings, including first-line, adjuvant, and neo-adjuvant therapy. In the scope of this review, we examined clinical studies utilizing ICIs in the context of treating oral mucosal melanoma, a rare disease, albeit with an extremely poor prognosis, with a specific focus on unraveling the intricate web of resistance mechanisms. The absence of a comprehensive review focusing on ICIs in oral mucosal melanoma is notable. Therefore, this review seeks to address this deficiency by offering a novel and thorough analysis of the current status, potential resistance mechanisms, and future prospects of applying ICIs specifically to oral malignant melanoma. Clarifying and thoroughly understanding these mechanisms will facilitate the advancement of effective therapeutic approaches and enhance the prospects for patients suffering from oral mucosal melanoma.

Characteristics of Sensory Neuron Dysfunction in Amyotrophic Lateral Sclerosis (ALS): Potential for ALS Therapy.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterised by the progressive degeneration of motor neurons, resulting in muscle weakness, paralysis, and, ultimately, death. Presently, no effective treatment for ALS has been established. Although motor neuron dysfunction is a hallmark of ALS, emerging evidence suggests that sensory neurons are also involved in the disease. In clinical research, 30% of patients with ALS had sensory symptoms and abnormal sensory nerve conduction studies in the lower extremities. Peroneal nerve biopsies show histological abnormalities in 90% of the patients. Preclinical research has reported several genetic abnormalities in the sensory neurons of animal models of ALS, as well as in motor neurons. Furthermore, the aggregation of misfolded proteins like TAR DNA-binding protein 43 has been reported in sensory neurons. This review aims to provide a comprehensive description of ALS-related sensory neuron dysfunction, focusing on its clinical changes and underlying mechanisms. Sensory neuron abnormalities in ALS are not limited to somatosensory issues; proprioceptive sensory neurons, such as MesV and DRG neurons, have been reported to form networks with motor neurons and may be involved in motor control. Despite receiving limited attention, sensory neuron abnormalities in ALS hold potential for new therapies targeting proprioceptive sensory neurons.

Oral frailty is a risk factor for body weight loss after gastrectomy: a single-center, retrospective study.

BACKGROUND: Body weight loss (BWL) is a serious complication of gastrectomy in patients with gastric cancer (GC). Nutritional intervention alone is inadequate for preventing BWL, and a new approach is needed. Oral frailty among older adults has recently attracted attention. This study aimed to investigate masticatory ability and BWL after gastrectomy. METHODS: This was a single-center, retrospective study. Functional tooth units (FTU) were used to measure masticatory ability. Patients with FTU < 4 were defined as low FTU group and FTU ≥ 4 as high FTU group. The BWL was compared between the two groups. RESULTS: Sixty patients who underwent distal gastrectomy for GC from March 2022 to January 2023 were enrolled in this study. The median FTU was 3 (range 0-12). The low-FTU group (FTU < 4) included 29 patients, while the high-FTU group (FTU ≥ 4) included 31 patients. The %BWL in the low FTU group was significantly higher than that in the high-FTU group at 1 and 3 months (p = 0.003 and p = 0.017, respectively). The risk factors associated with a %BWL > 5 at 1 and 3 months after gastrectomy were analyzed using logistic regression analysis. Only FTU < 4 was an independent risk factor after gastrectomy for GC in univariate and multivariate analyses (p = 0.028 and p = 0.006, respectively). CONCLUSIONS: Low FTU in patients with preoperative GC was a risk factor for %BWL 1 and 3 months postoperatively. Appropriate oral interventions may be useful in improving the postoperative nutritional status after gastrectomy.

Not brushing teeth at night may increase the risk of cardiovascular disease.

In this study, we investigated whether toothbrushing timing affects cardiovascular disease risk. We enrolled 1675 patients aged ≥ 20 years who were hospitalized for surgery, examination, or medical treatment. The participants were categorized as follows based on toothbrushing: Group MN (brushing teeth after waking up and at night, n = 409), Group Night (brushing teeth at night but not upon waking up, n = 751), Group M (brushing teeth after waking up but not at night, n = 164), and Group None (not brushing teeth at all, n = 259). The participants' age, sex, smoking history, and follow-up results were evaluated. Group M had four times as many men as women. Multivariate analysis of cardiovascular events showed significantly higher survival estimates in Group MN (P = 0.021) and Group Night (P = 0.004) than in Group None. Kaplan-Meier analysis of subgroups based on smoking status revealed that smokers in Group None had significantly worse prognosis for cardiovascular onset events than smokers in other groups; non-smokers in Groups None and M showed significantly worse prognosis on hospitalization. Our findings are limited to cardiovascular diseases and cannot be generalized to healthy populations. However, we suggest that brushing teeth at night is important for lowering cardiovascular disease risk.

TGF-ß generates a population of cancer cells residing in G1 phase with high motility and metastatic potential via KRTAP2-3.

Transforming growth factor ß (TGF-ß) increases epithelial cancer cell migration and metastasis by inducing epithelial-mesenchymal transition (EMT). TGF-ß also inhibits cell proliferation by inducing G1 phase cell-cycle arrest. However, the correlation between these tumor-promoting and -suppressing effects remains unclear. Here, we show that TGF-ß confers higher motility and metastatic ability to oral cancer cells in G1 phase. Mechanistically, keratin-associated protein 2-3 (KRTAP2-3) is a regulator of these dual effects of TGF-ß, and its expression is correlated with tumor progression in patients with head and neck cancer and migratory and metastatic potentials of oral cancer cells. Furthermore, single-cell RNA sequencing reveals that TGF-ß generates two populations of mesenchymal cancer cells with differential cell-cycle status through two distinctive EMT pathways mediated by Slug/HMGA2 and KRTAP2-3. Thus, TGF-ß-induced KRTAP2-3 orchestrates cancer cell proliferation and migration by inducing EMT, suggesting motile cancer cells arrested in G1 phase as a target to suppress metastasis.

LATS kinases and SLUG regulate the transition to advanced stage in aggressive oral cancer cells.

The epithelial-to-mesenchymal transition (EMT) is a critical process by which cancer cells acquire malignant features. However, the molecular mechanism and functional implications of EMT and the mesenchymal-to-epithelial transition (MET) in tumor progression remain elusive. In this study, we established two aggressive cancer cell lines from the human oral cancer cell line SAS, mesenchymal-like SAS-m4 and epithelial-like SAS-δ. SAS-δ is a revertant cell obtained by inducing MET in SAS-m4. SAS-δ, but not SAS-m4, exhibited abnormal cell growth, including piled-up overgrowth and invasive tumor formation in the tongues of nude mice, suggesting that SAS-δ represented more advanced cancer cells than the parental SAS cells. EMT-related transcriptional factor SLUG is phosphorylated at T208 and partly stabilized by the Hippo pathway kinases, LATS1 and LATS2. Depletion of SLUG promoted the invasive activity of SAS-δ by increasing the protein levels of LATS1/2 and the proportion of the phosphorylated form among total SLUG protein. Our results suggest that the LATS1/2-SLUG axis regulates the transition of SAS cells to the advanced stage via repeated switching between EMT and MET. Therefore, an anti-SLUG-pT208 antibody would be valuable not alone as a malignant tumor marker antibody but also as a prognostic tool for patients with malignant disease.

Ras signaling and RREB1 are required for the dissociation of medial edge epithelial cells in murine palatogenesis.

Cleft palate is one of the major congenital craniofacial birth defects. The etiology underlying the pathogenesis of cleft palate has yet to be fully elucidated. Dissociation of the medial edge epithelium (MEE) at the contacting region of palatal shelves and subsequent migration or apoptosis of MEE cells is required for proper MEE removal. Ras-responsive element-binding protein 1 (RREB1), a RAS transcriptional effector, has recently been shown to play a crucial role in developmental epithelial-mesenchymal transition (EMT), in which loss of epithelial characteristics is an initial step, during mid-gastrulation of embryonic development. Interestingly, the involvement of RREB1 in cleft palate has been indicated in humans. Here, we demonstrated that pan-Ras inhibitor prevents the dissociation of MEE during murine palatal fusion. Rreb1 is expressed in the palatal epithelium during palatal fusion, and knockdown of Rreb1 in palatal organ culture resulted in palatal fusion defects by inhibiting the dissociation of MEE cells. Our present findings provide evidence that RREB1-mediated Ras signaling is required during palatal fusion. Aberrant RREB1-mediated Ras signaling might be involved in the pathogenesis of cleft palate.

Oncolytic herpes virus G47Δ injected into tongue cancer swiftly traffics in lymphatics and suppresses metastasis.

The prognosis of oral squamous cell carcinoma (OSCC) largely depends on the control of lymph node metastases. We evaluate the therapeutic efficacy of G47Δ, a third-generation oncolytic herpes simplex virus type 1 (HSV-1), in mouse tongue cancer models. Intratumoral injection with G47Δ prolonged the survival in all orthotopic models investigated. In both athymic and immunocompetent models, G47Δ injected into the tongue cancer swiftly traffics to the draining cervical lymph nodes and suppresses lymph node metastases. In the immunocompetent KLN205-MUC1 model, in which the metastatic cascade that tongue cancer patients commonly experience is reproduced, intratumoral G47Δ injection even immediately prior to a tumor resection prolonged survival. Cervical lymph nodes 18 h after G47Δ treatment showed the presence of G47Δ infection and an increase in CD69-positive cells, indicating an immediate activation of T cells. Furthermore, G47Δ injected directly into enlarged metastatic lymph nodes significantly prolonged the survival at an advanced stage. Whereas intratumorally injected oncolytic HSV-1 does not readily circulate in the blood stream, G47Δ is shown to traffic in the lymphatics swiftly. The use of G47Δ can lead to entirely new treatment strategies for tongue cancer and other OSCC at all clinical stages.

Progression of melanoma is suppressed by targeting all transforming growth factor­ß isoforms with an Fc chimeric receptor.

Melanoma is an aggressive type of cancer originating from the skin that arises from neoplastic changes in melanocytes. Transforming growth factor­ß (TGF­ß) is a pleiotropic cytokine and is known to contribute to melanoma progression by inducing the epithelial­mesenchymal transition (EMT) program and creating an environment that favors tumor progression. There are three TGF­ß isoforms, TGF­ß1, TGF­ß2 and TGF­ß3, all of which engage in pro­tumorigenic activities by activating SMAD signaling pathways. All TGF­ß isoforms activate signaling pathways by binding to their TGF­ß type I (TßRI) and type II (TßRII) receptors. Thus, effective targeting of all TGF­ß isoforms is of great importance. In the present study, chimeric proteins comprising the extracellular domains of TßRI and/or TßRII fused with the Fc portion of human immunoglobulin (IgG) were validated in the melanoma context. The Fc chimeric receptor comprising both TßRI and TßRII (TßRI­TßRII­Fc) effectively trapped all TGF­ß isoforms. Conversely, TßRII­Fc chimeric receptor, that comprises TßRII only, was able to interact with TGF­ß1 and TGF­ß3 isoforms, but not with TGF­ß2, which is a poor prognostic factor for melanoma patients. Accordingly, it was revealed that TßRI­TßRII­Fc chimeric receptor suppressed the EMT program in melanoma cells in vitro induced by any of the three TGF­ß isoforms, as revealed by decreased expression of mesenchymal markers. Conversely, TßRII­Fc chimeric receptor inhibited the EMT program induced by TGF­ß1 and TGF­ß3. In addition, it was established that tumor growth in subcutaneous mouse melanoma was inhibited by TßRI­TßRII­Fc chimeric receptor indicating that Fc chimeric receptor could be applied to modify the tumor microenvironment (TME) of melanoma. Therefore, designing of Fc chimeric receptors targeting TGF­ß signals that affect various components of the TME may result in the development of effective anti­melanoma agents.

Salivary duct carcinoma of the submandibular gland presenting a diagnostic challenge: A case report.

BACKGROUND: Salivary duct carcinoma (SDC) is a rare, extremely aggressive malignancy that arises in the submandibular gland. It can metastasize locally early and therefore is an important differential diagnosis of metastatic disease in cervical lymph nodes or specific lymphadenitis such as tuberculous cervical lymphadenitis. CASE SUMMARY: We report a case of SDC in the submandibular gland that presented diagnostic difficulty. The lesion was coincidentally discovered through examination of the radiolucent area of the maxilla. Imaging failed to confirm the possibility of specific inflammation, leading us to execute an open biopsy to verify the diagnosis. The surgical specimen showed that the submandibular gland was primarily replaced with a calcified body. Following histological analysis and confirmation, we performed surgical resection, radiotherapy, and various chemotherapies. CONCLUSION: Radiographic imaging characteristics of lymph node metastases of salivary gland cancer, especially of SDC, may resemble other cervical lymphadenitis; calcification at the submandibular gland is the landmark of SDC occurring at the subman-dibular gland.

Primary Alveolar Soft Part Sarcoma of Cheek: Report of a Case and Review of the Literature.

Alveolar soft part sarcoma (ASPS) is a rare soft tissue sarcoma characterized by an alveolar or organoid arrangement of polygonal tumour cells separated by fibrovascular septa. A specific fusion gene [ASPS critical region 1 (ASPSCR1)-TFE3] was detected in ASPS. Despite being a slow-growing tumour without pain and dysfunction, ASPS is characterized by early metastasis, which leads to poor prognosis. Herein, we report a rare case of primary ASPS of the cheek harbouring ASPSCR1 (exon 7)-TFE3 (exon 5) fusion gene in a 21 year-old woman. This tumour was a well-circumscribed, smooth, round mass that was clinically suspected as a benign tumour. However, histologically, it was observed that the polygonal tumour cells were arranged in solid and alveolar growth patterns. Post-operative examination of the whole body excluded the possibility of metastasis at other sites. Thus, careful immunohistochemical and genetic analyses, as well as whole-body examination, demonstrated that the tumour was a primary ASPS of the cheek.

Clear Cell Carcinoma of Palatal Minor Salivary Gland Harboring a Novel EWSR1-ATF1 Fusion Gene: Report of a Case and Review of the Literature.

Clear cell carcinoma (CCC) is a rare low-grade malignant salivary gland carcinoma. EWSR1-ATF1 fusion has been characterized as a consistent finding in CCC, with breakpoints described between EWSR1 exon 11 and ATF1 exon 3. So far, over 100 cases of CCC harboring EWSR1 rearrangement arising from salivary gland of the oral cavity have been reported. Although EWSR1 involvement in these cases was confirmed by EWSR1 break-apart FISH indicating the translocation, sequence analysis for EWSR1-ATF1 fusion type has been reported only in three cases of CCC so far. Herein, we report a CCC case with novel EWSR1-ATF1 fusion (EWSR1 exon 15 and ATF1 exon 5) arising in minor salivary gland and review the role of the chimeric variants in some malignancies with EWSR1-ATF1 rearrangement. Current tumor was composed of the small nests of clear tumor cells and hyalized fibrous stroma. Immunohistochemically, the tumor was positive for AE1/AE3, CK5/6 and p63, negative for S100, Melan-A, SMA and CD10. After 8 months of follow-up, there are no evidence of recurrence.

Clathrin heavy chain phosphorylated at T606 plays a role in proper cell division.

Clathrin regulates mitotic progression, in addition to membrane trafficking. However, the detailed regulatory mechanisms of clathrin during mitosis remain elusive. Here, we demonstrate novel regulation of clathrin during mitotic phase of the cell cycle. Clathrin heavy chain (CHC) was phosphorylated at T606 by its association partner cyclin G-associated kinase (GAK). This phosphorylation was required for proper cell proliferation and tumor growth of cells implanted into nude mice. Immunofluorescence analysis showed that the localization of CHC-pT606 signals changed during mitosis. CHC-pT606 signals localized in the nucleus and at the centrosome during interphase, whereas CHC signals were mostly cytoplasmic. Co-immunoprecipitation suggested that CHC formed a complex with GAK and polo-like kinase 1 (PLK1). Depletion of GAK using siRNA induced metaphase arrest and aberrant localization of CHC-pT606, which abolished Kiz-pT379 (as a phosphorylation target of PLK1) signals on chromatin at metaphase. Taken together, we propose that the GAK_CHC-pT606_PLK1_Kiz-pT379 axis plays a role in proliferation of cancer cells.

Isolation of cancer cells with augmented spheroid-forming capability using a novel tool equipped with removable filter.

Three-dimensional (3D) cell culture systems have been used to obtain multicellular spheroidal cell aggregates, or spheroids, from cancer cells. However, it is difficult to efficiently prepare large tumor-derived spheroids from cancer cells. To circumvent this problem, we here used a tool equipped with removal membrane, called Spheroid Catch, for the selection and enrichment of large-sized and/or size-matched spheroids from human squamous cell carcinoma (SAS cells) without loss of recovery. After a five-round process of selection and enrichment, we successfully isolated a subpopulation of SAS cells with augmented spheroid-forming capability, named eSAS: the efficiency of spheroid formation is 28.5% (eSAS) vs 16.8% (parental SAS). Notably, we found that some of eSAS cells survived after exposure of high doses of cisplatin in 3D culture. Moreover, orthotopic implantation by injecting eSAS cells into the tongues of nude mice showed reduced survival rate and increased tumor growth compared with those of nude mice injected with SAS cells. These results suggest that spheroids exhibiting properties of higher spheroid forming capacity can be efficiently collected by using Spheroid Catch. Indeed, genome-wide cDNA microarray and western blot analyses demonstrated higher mRNA and protein levels of hedgehog acyltransferase (HHAT), which is associated with stem maintenance in cell carcinoma by catalysing the N-palmitoylation of Hedgehog proteins, in eSAS cells than in SAS cells. We propose that Spheroid Catch could be useful for the study of spheroids, and potentially organoids, in the basic and clinical sciences, as an alternative method to other type of cell strainers.

ELAS1 induces apoptotic death in adenocarcinoma DU145 and squamous-cell carcinoma SAS cancer cells, but not in normal KD cells.

We previously reported that an ELAS1 peptide containing 29 amino acids induces apoptotic death in U2OS human osteosarcoma cells following DNA double-strand break insults. Here, we show that ELAS1 also caused apoptosis in prostate adenocarcinoma DU145 cells and tongue squamous-cell carcinoma SAS cells. ELAS1 appears to be safe because it induced apoptosis only in cancer cells, not in normal KD cells. Because the effect of ELAS1 is dependent on increased stability of p53 and enhanced phosphorylation of p53-S46, we exogenously expressed wild-type p53 protein to fully promote ELAS1-mediated induction of apoptosis in SAS cells. Interestingly, simultaneous expression of Myc-ELAS1 and FLAG-p53 mediated by an internal ribosome entry site efficiently induced apoptosis in SAS cells. Moreover, we prepared a recombinant adenovirus that simultaneously expressed Myc-ELAS1 and FLAG-p53. This adenovirus also killed SAS cells, as determined by a cell viability assay, in the presence of camptothecin, an inducer of DNA double-strand breaks. Moreover, nude mice harboring Myc-ELAS1-expressing SAS cells lived longer than mice harboring Myc-vector-expressing SAS cells, suggesting the usefulness of ELAS1 in vivo. Notably, Cy5-tagged ELAS1-t, which contained only ten amino acids, also efficiently induced apoptosis in both DU145 and SAS cells, suggesting the usefulness of ELAS1-t as a peptide. Taken together, our results suggest that ELAS1 is therapeutically useful as a peptide drug.

GAK is phosphorylated by c-Src and translocated from the centrosome to chromatin at the end of telophase.

Cyclin G-associated kinase (GAK) harbors a consensus phosphorylation motif (Y412) for c-Src; however, its physiological significance remains elusive. Here, we show that GAK is phosphorylated by c-Src not only at Y412 but also at Y1149. An anti-GAK-pY412 antibody recognized the shifted band of GAK during M phase. Immunofluorescence (IF) showed that GAK-pY412/pY1149 signals were present in the nucleus during interphase, translocated to chromosomes at prophase and prometaphase, moved to centrosomes at metaphase, and finally translocated to chromosomes at the end of telophase, when nuclear membrane formation was almost complete. These subcellular movements of GAK resemble those of DNA licensing factors. Indeed, mass spectrometry identified mini-chromosome maintenance (MCM) 3, an essential component of the DNA licensing system, as one of the association partners of GAK; immunoprecipitation-mediated Western blotting confirmed their association in vivo. These results suggest that the c-Src_GAK_MCM axis plays an important role in cell cycle progression through control of the DNA replication licensing system.

Comprehensive phenotypic analysis of knockout mice deficient in cyclin G1 and cyclin G2.

Cyclin G1 (CycG1) and Cyclin G2 (CycG2) play similar roles during the DNA damage response (DDR), but their detailed roles remain elusive. To investigate their distinct roles, we generated knockout mice deficient in CycG1 (G1KO) or CycG2 (G2KO), as well as double knockout mice (DKO) deficient in both proteins. All knockouts developed normally and were fertile. Generation of mouse embryonic fibroblasts (MEFs) from these mice revealed that G2KO MEFs, but not G1KO or DKO MEFs, were resistant to DNA damage insults caused by camptothecin and ionizing radiation (IR) and underwent cell cycle arrest. CycG2, but not CycG1, co-localized with γH2AX foci in the nucleus after γ-IR, and γH2AX-mediated DNA repair and dephosphorylation of CHK2 were delayed in G2KO MEFs. H2AX associated with CycG1, CycG2, and protein phosphatase 2A (PP2A), suggesting that γH2AX affects the function of PP2A via direct interaction with its B'γ subunit. Furthermore, expression of CycG2, but not CycG1, was abnormal in various cancer cell lines. Kaplan-Meier curves based on TCGA data disclosed that head and neck cancer patients with reduced CycG2 expression have poorer clinical prognoses. Taken together, our data suggest that reduced CycG2 expression could be useful as a novel prognostic marker of cancer.