As the leading cancer of the female reproductive tract, it is not uncommon for human papilloma virus (HPV)-associated cervical squamous cell carcinoma (HPV-CSCC) to metastasize to pelvic organs and lymph nodes in advanced stages. However, herein, we present a rare case in which superficial invasive HPV-CSCC metastasized to the unilateral ovary as a large mass by spreading directly through the endometrium and fallopian tubes and lymph-vascular space invasion. The case is so unexpected that the misdiagnosis most likely could be proceeded as a primary ovarian cancer.A 58-year-old postmenopausal woman presented vaginal bleeding for more than 4 months, never received hormonal treatment and had no family history of malignant diseases. Routine ultrasound revealed a 12 × 10 × 10 cm right ovarian mass. Intraoperative frozen section was diagnosed as a borderline Brenner tumour with local highly suspected invasive carcinoma. Accordingly, omentectomy surgery then occurred. Unbelievably, by observation under a microscope, immunohistochemistrial staining, and HPV RNA scope, we found that the carcinoma originated from the uterine cervix. In the uterine cervix, stage IA1 superficial invasive squamous carcinoma was found, and the carcinoma directly spread to the endometrium and bilateral fallopian tube, was planted into the right ovary and eventually grew as a large mass. Moreover, lymph-vascular space invasion (LVSI) was also discovered. To date, the patient has been given 6 cycles of chemotherapy and has experienced no recurrence.The diagnosis of superficial invasive cervical squamous cell carcinoma metastasizing to the ovary is very challenging for pathological doctors, especially in intraoperative consultations.

Neurotrophic herpes simplex virus type 1 (HSV-1) establishes lifelong latent infection in humans. Accumulating studies indicate that HSV-1, a risk factor of neurodegenerative diseases, exacerbates the sporadic Alzheimer's disease (AD). The analysis of viral genetic materials via genomic sequencing and quantitative PCR (qPCR) is the current approach used for the detection of HSV-1; however, this approach is limited because of its difficulty in detecting both latent and lytic phases of the HSV-1 life cycle in infected hosts. RNAscope, a novel in situ RNA hybridization assay, enables visualized detection of multiple RNA targets on tissue sections. Here, we developed a fluorescent multiplex RNAscope assay in combination with immunofluorescence to detect neuronal HSV-1 transcripts in various types of mouse brain samples and human brain tissues. Specifically, the RNA probes were designed to separately recognize two transcripts in the same brain section: (1) the HSV-1 latency-associated transcript (LAT) and (2) the lytic-associated transcript, the tegument protein gene of the unique long region 37 (UL37). As a result, both LAT and UL37 signals were detectable in neurons in the hippocampus and trigeminal ganglia (TG). The quantifications of HSV-1 transcripts in the TG and CNS neurons are correlated with the viral loads during lytic and latent infection. Collectively, the development of combinational detection of neuronal HSV-1 transcripts in mouse brains can serve as a valuable tool to visualize HSV-1 infection phases in various types of samples from AD patients and facilitate our understanding of the infectious origin of neurodegeneration and dementia.

The homeodomain transcription factor SIX3 is a known regulator of eye, nose, and forebrain development, and has recently been implicated in female reproduction. Germline heterozygosity of SIX3 is sufficient to cause subfertility, but the cell populations that mediate this role are unknown. The neuropeptide kisspeptin is a critical component of the reproductive axis and plays roles in sexual maturation, ovulation, and the maintenance of gonadotropin secretion. We used Cre-Lox technology to remove Six3 specifically from kisspeptin neurons in mice to test the hypothesis that SIX3 in kisspeptin neurons is required for reproduction. We found that loss of Six3 in kisspeptin neurons causes subfertility and estrous cycle irregularities in females, but no effect in males. Overall, we find that SIX3 expression in kisspeptin neurons is an important contributor to female fertility.

The neuropeptide oxytocin (Oxt) plays important roles in modulating social behaviors. Oxt receptor (Oxtr) is abundantly expressed in the brain and its relationship to socio-behavioral controls has been extensively studied using mouse brains. Several genetic tools to visualize and/or manipulate Oxtr-expressing cells, such as fluorescent reporters and Cre recombinase drivers, have been generated by ES-cell based gene targeting or bacterial artificial chromosome (BAC) transgenesis. However, these mouse lines displayed some differences in their Oxtr expression profiles probably because of the complex context and integrity of their genomic configurations in each line. Here, we apply our sophisticated genome-editing techniques to the Oxtr locus, systematically generating a series of knock-in mouse lines, in which its endogenous transcriptional regulations are intactly preserved and evaluate their expression profiles to ensure the reliability of our new tools. We employ the epitope tagging strategy, with which C-terminally fused tags can be detected by highly specific antibodies, to successfully visualize the Oxtr protein distribution on the neural membrane with super-resolution imaging for the first time. By using T2A self-cleaving peptide sequences, we also induce proper expressions of tdTomato reporter, codon-improved Cre recombinase (iCre), and spatiotemporally inducible Cre-ERT2 in Oxtr-expressing neurons. Electrophysiological recordings from tdTomato-positive cells in the reporter mice support the validity of our tool design. Retro-orbital injections of AAV-PHP.eB vector into the Cre line further enabled visualization of recombinase activities in the appropriate brain regions. Moreover, the first-time Cre-ERT2 line drives Cre-mediated recombination in a spatiotemporally controlled manner on tamoxifen (TMX) administration. These tools thus provide an excellent resource for future functional studies in Oxt-responsive neurons and should prove of broad interest in the field.

To determine whether TGFB1 affects the immune microenvironment of ccRCC, we investigated the association between TGFB1 expression and clinicopathological features. Tissue microarray was generated from 158 total or partial nephrectomy samples and 12 tumor-adjacent normal kidney tissue. TGFB1 expression was assessed by RNA in situ hybridization and quantified using ImageJ software. TGFB1 was significantly upregulated in ccRCC tissue than in normal kidney tissues (P = 1.03 × 10-9). Tumors with a high WHO/ISUP grade had higher TGFB1 expression levels (P = 7.05 × 10-3). Of 139 patients with localized ccRCC and whose follow-up data were available, those in the TGFB1-high group displayed significantly shorter relapse-free survival than those in the TGFB1-low group (P = 0.0251). TGFB1 expression was significantly upregulated in patients who developed distant metastasis after surgery (n = 12) than in patients without metastasis (n = 127; P = 0.00167). TGFB1 expression positively correlated with the number of PD-L1-positive cells in the tumor stroma (P = 0.0206, ρ = 0.163). Furthermore, TGFB1 expression was associated with the formation of tertiary lymphoid structures. TGF-β1 is a prognostic indicator of worse outcome for ccRCC and might be a therapeutic target in advanced ccRCC. Our data provide new insights into the association between tumor biology and tumor microenvironment in ccRCC.

Post-mortem examination plays a pivotal role in understanding the pathobiology of the SARS-CoV-2; thus, the optimization of virus detection on the post-mortem formalin-fixed paraffin-embedded (FFPE) tissue is needed. Different techniques are available for the identification of the SARS-CoV-2, including reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy. The main goal of this study is to compare ISH versus RT-PCR to detect SARS-CoV-2 on post-mortem lung samples of positive deceased subjects. A total of 27 samples were analyzed by RT-PCR targeting different viral RNA sequences of SARS-CoV-2, including envelope (E), nucleocapsid (N), spike (S), and open reading frame (ORF1ab) genes and ISH targeting S and Orf1ab. All 27 cases showed the N gene amplification, 22 out of 27 the E gene amplification, 26 out of 27 the S gene amplification, and only 6 the ORF1ab gene amplification. The S ISH was positive only in 12 out of 26 cases positive by RT-PCR. The S ISH positive cases with strong and diffuse staining showed a correlation with low values of the number of the amplification cycles by S RT-PCR suggesting that ISH is a sensitive assay mainly in cases carrying high levels of S RNA. In conclusion, our findings demonstrated that ISH assay has lower sensitivity to detect SARS-CoV-2 in FFPE compared to RT-PCR; however, it is able to localize the virus in the cellular context since it preserves the morphology.

Mutations in proteins forming the contractile apparatus of cardiac muscle can alter muscle function, leading to a hypercontractile or a hypocontractile state of the heart. These mutational insults can also lead to secondary cardiac remodeling and disease states over time, resulting in either hypertrophic cardiomyopathy (HCM), affecting about 1 in 500 individuals, or dilated cardiomyopathy (DCM), affecting about 1 in 250 individuals. We are applying a broad range of techniques to follow the initial atomic-level insults to tissue-level remodeling and function in an effort to better understand mechanisms of disease. Here, we characterize four variants of unknown significance in tropomyosin-1 (Tpm1.1), a key regulatory protein on muscle thin filaments, using a combination of molecular modeling techniques, in vitro motility assays, and analysis in whole tissue derived from engineered induced-pluripotent stem cells. First, a list of about 20 variants of unknown significance were analyzed computationally using molecular dynamics and energy minimization calculations to predict each mutant’s effects on Tpm1.1 structure and association with thin filament proteins. From this analysis, four mutations (A102D, D258E, K233N, and A239T) were selected for further study. A multiscale model of myofilament activation was then used to synthesize key parameters distilled from atomistic simulations, allowing ab initio predictions of the impact of each variant on myofilament calcium sensitivity and twitch force phenotype. These predictions were then tested for each variant via in vitro motility assays and human engineered heart tissues virally expressing mutant Tpm1.1. A comparison between predicted and actual variant phenotypes reveals robust progress toward our long-term goal of computational prediction of disease risk for novel Tpm1.1 variants while simultaneously highlighting new challenges and opportunities with this method.

Calmodulin (CaM) is a multifunctional calcium-binding protein that modulates activity of many different ion channels, enzymes and other proteins. In vertebrates, CaM represents a unique case, where the exact same, invariant amino acid sequence is encoded by multiple genes on different chromosomes (three in case of the mouse and human). These multiple CaM genes have been hypothesized to afford high spatiotemporal resolution in the control of numerous CaM-dependent processes within highly specialized cells, such as neurons and cardiac myocytes. Single molecule detection of mRNAs transcribed from Calm1, Calm2 and Calm3 genes in cardiac myocytes (RNAScope) revealed significantly different spatial distributions within murine ventricular cardiomyocytes for the three mRNAs: 1) Calm2 mRNA was concentrated in the cell core (closer to the nuclei than the external plasma membrane); 2) Calm3mRNA was more concentrated along the myocyte periphery; whereas 3) the mRNA for Calm1 is more concentrated between the Calm2 and Calm3 densities. We further found that Calm2 mRNA (but not Calm1 and Calm3) exhibited a significant overlap with mRNA of the ryanodine receptor Ca2þ release channel (Ryr2). Moreover, in vivo sympathetic augmentation by isoproterenol infusion from implanted osmotic mini-pumps resulted in both marked upregulation of Calm3 mRNA (but not Calm2 and Calm1) and enhanced overlap between Calm3 and Ryr2 mRNA mRNA at intercalated discs. These results suggest that spatial and temporal organization of CaM-Ca2þ signaling in cardiac myocytes involves geometrically and functionally diverse CaM pools. Functionally distinct CaM pools are maintained through co-translation of Calm mRNAs with specific target protein mRNAs, such as Ryr2, and their modulation by neurotransmitters, such as adrenergic agonists.

Cardiac myosin-binding protein C (cMyBP-C) is an important regulator of cardiac muscle contraction and is commonly implicated in hypertrophic cardiomyopathy (HCM). However, the mechanism of regulation by cMyBP-C remains unclear due to experimental challenges in dissecting the proposed weak, transient interactions with its binding partners. Here, we utilized a nanosurf assay, containing a synthetic b-cardiac myosin thick filament, to systematically probe cMyBP-C interactions with actin and myosin. Recombinant human b-cardiac myosin subfragments (HMM or S1) were attached to DNA nanotubes, with 14 or 28 nm spacing, similar to the myosin head spacing on native thick filaments. No significant difference in thin filament velocity was observed with 14 nm vs. 28 nm motor spacing. Various N-terminal fragments of cMyBP-C were interdigitated with b-cardiac myosin on DNA nanotubes via encoded SNAP tags labeled with sequence-specific oligo attachment strands. We recapitulated inhibition of thin filament motility on b-cardiac myosin HMM and S1 nanotubes by C0-C2 (4-6 fold) and C1-C2 (4-8 fold) N-terminal cMyBP-C fragments. Equivalent inhibition of b-cardiac myosin HMM and S1 subfragments suggests the actin-cMyBP-C interaction dominates this inhibitory mechanism. We found that a C0-C1f fragment lacking the majority of the M-domain did not inhibit b-cardiac myosin nanotube motility, confirming the importance of the M-domain in regulatory interactions. Diminished inhibition by C0-C2 and C1-C2 phosphomimetic fragments (2-3 fold higher velocity compared to their phosphonull counterparts) further highlights the importance of the phosphorylatable serines in the regulatory Mdomain. These results shed light on the mechanism of cMyBP-C and highlight the utility of the nanosurf as

Cardiac troponin T (cTnT) is a protein of the cardiac thin filament (CTF) and assists in conferring calcium regulation to muscle contraction. Mutations in cTnT often cause hypertrophic cardiomyopathy (HCM), a disease affecting 1/500 people worldwide. This study focuses on six HCM-causing, highly penetrant mutations located within the cTnT N-terminus (R94H/C, R92L/W/Q, and I79N) which are each associated with distinct phenotypes and severities in human patients. The goal of this study was to determine the effects of HCMcausing mutations in cTnT on the calcium-based regulation of muscle activation. Using fluorescently labeled, bacterially expressed, recombinant human protein, we measured in vitro calcium exchange (sensitivity via spectrofluorimetry and kinetics via stopped-flow) of human cTn and CTF complexes in the presence and absence of these disease-causing mutations. Disease-causing HCM mutations in cTn complexes alone resulted in no significant changes in either calcium sensitivity or calcium dissociation kinetics compared to wildtype (WT) controls. Alternatively, in the CTF every mutation significantly sensitized TnC to calcium. These results indicate that actin and tropomyosin are necessary to observe the effects of mutations on CTF activation. Although all mutations significantly increased calcium sensitivity of CTFs, four mutations (R92L/Q and R94H/C) significantly decreased the rate of calcium dissociation (1.2-1.5 fold), whereas two mutations significantly accelerated calcium dissociation (1.1-1.4 fold). Three mutations significantly accelerated calcium association (R92W, I79N, and R94C) 2.8-4.5 fold while a fourth trended with a slight, albeit functionally significant acceleration (R94H) at 2.0 fold. Thus, the calcium sensitization reported here for each mutation is accomplished via mutation-specific changes to the kinetics of calcium exchange with TnC. Furthermore, these results suggest that the kinetics of calcium exchange with TnC in the CTF system afford high resolution, mutation-specific mechanistic insight into altered myofilament calcium sensitivity that may ultimately facilitate targeted interventions.

Given the recent success of gene therapy modalities and the growing number of cell and gene-based therapies in clinical development across many different therapeutic areas, it is evident that this evolving field holds great promise for the unmet medical needs of patients. The recent approvals of Luxturna and Zolgensma prove that recombinant adeno-associated virus (rAAV)-based gene therapy is a transformative modality that enables curative treatment for genetic disorders. Over the last decade, Takeda has accumulated significant experience with rAAV-based gene therapies, especially in the early stage of development. In this review, based on the learnings from Takeda and publicly available information, we aim to provide a guiding perspective on Drug Metabolism and Pharmacokinetics (DMPK) substantial role in advancing therapeutic gene therapy modalities from nonclinical research to clinical development, in particular the characterization of gene therapy product biodistribution, elimination (shedding), immunogenicity assessment, multiple platform bioanalytical assays, and first-in-human (FIH) dose projection strategies. Graphical abstract.

Bovine respiratory disease (BRD) is the most significant cause of cattle morbidity and mortality worldwide. This multifactorial disease has a complex aetiology. Dogma posits a primary viral infection followed by secondary bacterial pneumonia. Bovine rhinitis B virus (BRBV) is an established aetiological agent of BRD, but little is known regarding its pathogenesis. Here, a BRD PCR panel identified 18/153 (11.8 %) lung samples and 20/49 (40.8 %) nasal swabs collected from cattle with respiratory signs as positive for BRBV, which was the most prevalent virus in nasal swabs. Primary bovine tracheal epithelial cells were used to isolate BRBV that was phylogenetically related to contemporary sequences from the USA and Mexico and genetically divergent from the previous sole BRBV isolate. To investigate virus pathogenesis, 1-week-old colostrum-deprived dairy calves were inoculated intranasally with 7.0 log10 TCID50 BRBV. Virus was isolated from nasal swabs, nasal turbinates, trachea and the brain of the challenged animals. Neutralizing antibodies were detected beginning 7 days post-inoculation and peaked at day 14. In situ hybridization (ISH) localized BRBV infection in the upper respiratory ciliated epithelial and goblet cells, occasionally associated with small defects of the superficial cilia lining. Sporadically, pinpoint ISH signals were also detected in cells resembling glial cells in the cerebrum in one calf. Together, these results demonstrate the BRBV infection is highly prevalent in acute BRD samples and while the pathogenicity of BRBV is minimal with infection largely limited to the upper respiratory tract, further research is needed to elucidate a possible initiatory role in BRD.