Spatiotemporal Control of Protein Refolding through Flash-Change Reaction Conditions.

Recombinant protein production is an essential aspect of biopharmaceutical manufacturing, with Escherichia coli serving as a primary host organism. Protein refolding is vital for protein production; however, conventional refolding methods face challenges such as scale-up limitations and difficulties in controlling protein conformational changes on a millisecond scale. In this study, we demonstrate the novel application of flow microreactors (FMR) in controlling protein conformational changes on a millisecond scale, enabling efficient refolding processes and opening up new avenues in the science of FMR technology. FMR technology has been primarily employed for small-molecule synthesis, but our novel approach successfully expands its application to protein refolding, offering precise control of the buffer pH and solvent content. Using interleukin-6 as a model, the system yielded an impressive 96% pure refolded protein and allowed for gram-scale production. This FMR system allows flash changes in the reaction conditions, effectively circumventing protein aggregation during refolding. To the best of our knowledge, this is the first study to use FMR for protein refolding, which offers a more efficient and scalable method for protein production. The study results highlight the utility of the FMR as a high-throughput screening tool for streamlined scale-up and emphasize the importance of understanding and controlling intermediates in the refolding process. The FMR technique offers a promising approach for enhancing protein refolding efficiency and has demonstrated its potential in streamlining the process from laboratory-scale research to industrial-scale production, making it a game-changing technology in the field.

Association of preconception dysmenorrhea with obstetric complications: the Japan Environment and Children's Study.

BACKGROUND: The association of maternal preconception dysmenorrhea, especially primary dysmenorrhea, with obstetric complications has not been clearly described. Therefore, we evaluated the association of preconception dysmenorrhea with obstetric complications while accounting for the presence of pelvic pathologies. METHODS: We analyzed the data of women with singleton live births at and after 22 weeks of gestation enrolled in the Japan Environment and Children's Study, a nationwide birth cohort study, between 2011 and 2014. Participants with psychological disorders were excluded. Preconception dysmenorrhea, identified in the medical record transcripts, was categorized into mild dysmenorrhea (MD) and severe dysmenorrhea (SD). Furthermore, excluding those who had pelvic pathologies via self-reported questionnaires (endometriosis, adenomyosis, and uterine myomas) with MD and SD, preconception dysmenorrhea was categorized into mild primary dysmenorrhea (MPD) and severe primary dysmenorrhea (SPD), respectively. Using multiple logistic regression, adjusted odds ratios (aORs) for obstetric complications, including preterm birth (PTB) before 37 weeks and 34 weeks, small-for-gestational-age infants, preterm premature rupture of membrane, and hypertensive disorders of pregnancy, were calculated (considering confounders) in women with (1) MD or SD and (2) MPD or SPD. Women without preconception dysmenorrhea were used as a reference. RESULTS: A total of 80,242 participants were analyzed. In women with SD, the aOR for PTB before 37 weeks was 1.38 (95% confidence interval [CI] 1.10, 1.72). In women with SPD, the aOR for PTB before 37 weeks was 1.32 (95% CI 1.02, 1.71). There was no association between women with MD or MPD and obstetric complications. CONCLUSIONS: SD and SPD are significantly associated with an increased incidence of PTB before 37 weeks. Care providers should provide proper counseling regarding the association between preconception dysmenorrhea and obstetric complications. Optimal management of pregnant women with preconception dysmenorrhea to reduce the incidence of PTB should be elucidated in further studies, with detailed clinical data of pelvic pathologies.

Rare Hereditary Gynecological Cancer Syndromes.

Hereditary cancer syndromes, which are characterized by onset at an early age and an increased risk of developing certain tumors, are caused by germline pathogenic variants in tumor suppressor genes and are mostly inherited in an autosomal dominant manner. Therefore, hereditary cancer syndromes have been used as powerful models to identify and characterize susceptibility genes associated with cancer. Furthermore, clarification of the association between genotypes and phenotypes in one disease has provided insights into the etiology of other seemingly different diseases. Molecular genetic discoveries from the study of hereditary cancer syndrome have not only changed the methods of diagnosis and management, but have also shed light on the molecular regulatory pathways that are important in the development and treatment of sporadic tumors. The main cancer susceptibility syndromes that involve gynecologic cancers include hereditary breast and ovarian cancer syndrome as well as Lynch syndrome. However, in addition to these two hereditary cancer syndromes, there are several other hereditary syndromes associated with gynecologic cancers. In the present review, we provide an overview of the clinical features, and discuss the molecular genetics, of four rare hereditary gynecological cancer syndromes; Cowden syndrome, Peutz-Jeghers syndrome, DICER1 syndrome and rhabdoid tumor predisposition syndrome 2.

Successful laparoscopic resection of ovarian abscess caused by Staphylococcus aureus in a 13-year-old girl: a case report and review of literature.

BACKGROUND: Ovarian abscesses, which occur mostly in sexually active women via recurrent salpingitis, occur rarely in virginal adolescent girls. Here, we present a case of an ovarian abscess in a virginal adolescent girl who was diagnosed and treated by laparoscopy. CASE PRESENTATION: A 13-year-old healthy girl presented with fever lasting for a month without abdominal pain. Computed tomography scan and magnetic resonance imaging indicated a right ovarian abscess. Laparoscopic surgery revealed a right ovarian abscess with intact uterus and fallopian tubes. The abscess was caused by Staphylococcus aureus. The patient recovered completely after excision of the abscess, followed by antibiotic treatment. CONCLUSIONS: Ovarian abscess may occur in virginal adolescent girls; Staphylococcus aureus, an uncommon species causing ovarian abscess, may cause the infection.

Reduced Claudin-12 Expression Predicts Poor Prognosis in Cervical Cancer.

BACKGROUND: Within the claudin (CLDN) family, CLDN12 mRNA expression is altered in various types of cancer, but its clinicopathological relevance has yet to be established due to the absence of specific antibodies (Abs) with broad applications. METHODS: We generated a monoclonal Ab (mAb) against human/mouse CLDN12 and verified its specificity. By performing immunohistochemical staining and semiquantification, we evaluated the relationship between CLDN12 expression and clinicopathological parameters in tissues from 138 cases of cervical cancer. RESULTS: Western blot and immunohistochemical analyses revealed that the established mAb selectively recognized the CLDN12 protein. Twenty six of the 138 cases (18.8%) showed low CLDN12 expression, and the disease-specific survival (DSS) and recurrence-free survival rates were significantly decreased compared with those in the high CLDN12 expression group. We also demonstrated, via univariable and multivariable analyses, that the low CLDN12 expression represents a significant prognostic factor for the DSS of cervical cancer patients (HR 3.412, p = 0.002 and HR 2.615, p = 0.029, respectively). CONCLUSIONS: It can be concluded that a reduced CLDN12 expression predicts a poor outcome for cervical cancer. The novel anti-CLDN12 mAb could be a valuable tool to evaluate the biological relevance of the CLDN12 expression in diverse cancer types and other diseases.

Successful management of a submucosal fibroid using a hysteroscopic morcellator system in a patient with a history of total proctocolectomy: A case report.

Transcervical resection is widely used in the management of submucosal fibroids. However, uterine perforation and thermal bowel injuries are known complications associated with the procedure. This is a case report of a 44-year-old woman who presented with heavy menstrual bleeding and anemia. Magnetic resonance imaging and hysteroscopy revealed multiple fibroids, including a submucosal type 1 fibroid. She had previously undergone a total proctocolectomy with intestinal pouch-anal anastomosis for ulcerative colitis. Because there was a high risk of permanent colostomy in the event of a bowel injury, an electrode-free, operative hysteroscopy using the Intrauterine Bigatti Shaver (IBS), a hysteroscopic morcellator system, was employed to prevent thermal bowel injury. The fibroid was completely removed using the IBS system with no complications. Therefore, we recommend the electrode-free use of a hysteroscopic morcellator system in patients with a history of extensive pelvic surgery.

Sucupiranins A-L, Furanocassane Diterpenoids from the Seeds of Bowdichia virgilioides.

Twelve new furanocassane diterpenoids, sucupiranins A-L (1-12), and three known compounds (13-15) were isolated from the seeds of Bowdichia virgilioides. The structures of the compounds were elucidated via 1H and 13C NMR analysis, including 2D NMR (1H-1H COSY, HSQC, HMBC, and NOESY); HRMS data; and X-ray crystallographic analysis. The absolute configurations were defined using their electronic circular dichroism (ECD) spectra by applying the exciton chirality method to the bis-p-bromobenzoate of compound 13. Sucupiranin J (10) inhibited lipopolysaccharide-induced nitric oxide production (IC50 30.6 µM), whereas sucupiranins J (10), K (11), and 13 exhibited weak antimalarial activity against Plasmodium falciparum K1 with IC50 values of 32.2, 23.5, and 22.9 µM and selectivity indices of 4.3, 1.9, and >12.0 (MRC-5/K1), respectively.

Successful management of locally advanced cervical cancer via total pelvic exenteration following neoadjuvant chemotherapy.

Stage IVA cervical cancer is a tumor that invades the mucosa of the bladder or rectum without distant metastasis and is difficult to treat, and concurrent chemoradiotherapy is recommended. Although radical surgery following neoadjuvant chemotherapy is a treatment option for stage IVA cervical cancer, the evidence is limited. A 51-year-old woman with bulky cervical cancer and rectal invasion was referred to our hospital. Paclitaxel and cisplatin were administered as neoadjuvant chemotherapies. After two cycles of chemotherapy, the tumor size decreased markedly. Total pelvic exenteration was performed, and a complete resection was achieved. Four cycles of paclitaxel and cisplatin were administered postoperatively. Thirty-three months after the completion of adjuvant chemotherapy, the patient was alive and free of disease. Radical surgery after neoadjuvant chemotherapy may be a treatment option for stage IVA cervical cancer with bulky tumors.

Successful Management of Ovarian Cancer Progressing on Olaparib by Niraparib Following Cytoreduction: A Case Report.

BACKGROUND: Polyadenosine 5'-diphosphoribose polymerase inhibitors (PARP-Is) are novel, effective agents for treating newly diagnosed epithelial ovarian cancer (EOC). However, the effect of PARP-I on the progression of recurrent EOC has not yet been determined. In particular, there is limited evidence regarding retreatment with PARP-I for recurrent EOC that has progressed on PARP-I in the short term. CASE REPORT: A 69-year-old woman with a BRCA1 mutated EOC relapsed five months after starting olaparib maintenance following neoadjuvant chemotherapy and interval debulking surgery. Although the platinum-free interval was within six months, secondary cytoreductive surgery was performed because the tumor was locoregional. Following two cycles of weekly nedaplatin, niraparib induced a complete response, and the patient maintained a progression-free status for 15 months. CONCLUSION: Even with short-term progression on PARP-I, local control combined with different platinum agents and PARP-I can be used to achieve good responses.

Diaphragmatic clear cell carcinoma with Lynch syndrome after surgery for atypical endometrial hyperplasia and ovarian endometriosis: A case report.

Clear cell carcinoma (CCC) of the diaphragm is rare, with an origin that is reported to be associated with malignant transformation of extraperitoneal endometriosis. Lynch syndrome (LS) is an autosomal dominant hereditary cancer syndrome caused by germline pathogenic variants in one of the DNA mismatch repair (MMR) genes, MLH1, MSH2, MSH6 and PMS2. Women with LS have a significantly increased lifetime risk of endometrial and ovarian cancer. CCC is a common histology of endometriosis- and LS-associated malignancy. The present study describes the case of a 51-year-old woman with an intra-abdominal mass found during a routine physical examination. The patient had undergone total hysterectomy and bilateral adnexectomy for atypical endometrial hyperplasia (AEH) and ovarian endometriosis, respectively, 3 years previously. Enhanced computed tomography showed a mass on the surface of the liver. Laparoscopic examination of the abdominal cavity revealed a tumor on the underside of the right diaphragm, which was then surgically excised. Pathological examination of the excised tumor, along with immunohistochemistry, led to a diagnosis of CCC. Since LS was suspected due to the genetic family history of the patient, microsatellite instability analysis was performed on the diaphragmatic tumor, and the results were positive. Immunohistochemistry was performed for MMR proteins in AEH and CCC cells, both of which revealed loss of MSH2 and MSH6 expression. Following detailed genetic counseling, genetic testing of MMR genes was performed, revealing a germline pathogenic variant in MSH2 (c.1000C>T, p.Gln344*), thus confirming the diagnosis of LS. To the best of our knowledge, this is the first case report of concurrent diaphragmatic CCC and LS. Patients with LS and endometriosis are at risk of developing ovarian cancer or intra-abdominal malignant tumors. In addition, immunohistochemistry screening for MMR proteins should be considered in patients with AEH and a family history of LS-related cancer, to enable early clinical intervention in cases of endometrial cancer.

AJICAP-M: Traceless Affinity Peptide Mediated Conjugation Technology for Site-Selective Antibody-Drug Conjugate Synthesis.

A traceless site-selective conjugation method, "AJICAP-M", was developed for native antibodies at sites using Fc-affinity peptides, focusing on Lys248 or Lys288. It produces antibody-drug conjugates (ADCs) with consistent drug-to-antibody ratios, enhanced stability, and simplified manufacturing. Comparative in vivo assessment demonstrated AJICAP-M's superior stability over traditional ADCs. This technology has been successfully applied to continuous-flow manufacturing, marking the first achievement in site-selective ADC production. This manuscript outlines AJICAP-M's methodology and its effectiveness in ADC production.

Construction Protocol of Drug-Protein Cage Complexes for Drug Delivery System.

Ferritin is one of the most promising drug delivery system (DDS) carriers because of its uniform nanosize, biodistribution, efficient cellular uptake, and biocompatibility. Conventionally, a disassembly/reassembly method that requires pH change has been used for the encapsulation of molecules in ferritin protein nanocages. Recently, a one-step method in which a complex of ferritin and a targeted drug was obtained by incubating the mixture at an appropriate pH, was established. Here, we describe two types of protocols, the conventional disassembly/reassembly method, and the novel one-step method for the construction of a ferritin-encapsulated drug using doxorubicin as an example molecule.

Using Dictionary Matching to Improve the Accuracy of MOLLI Myocardial T1 Analysis and Measurements of Heart Rate Variability.

We analyzed modified Look-Locker inversion recovery (MOLLI) T1 measurements by applying a dictionary matching strategy and aimed to acquire T1 measurements more accurately than those acquired by the conventional three-parameter matching analysis. We particularly clarified the robustness of this method for measuring heart rate (HR) variability. A phantom experiment using a 3T MRI system was performed for various HRs. The ideal MOLLI signal corresponding to the scan parameter in the MRI experiment was simulated over a wide range of T1 values according to the dictionary. The unknown T1 values were determined by finding the simulated signals in the dictionary corresponding to the measured signals using pattern matching. The measured T1 values showed that the proposed analysis improved the accuracy of T1 measurements compared to those acquired by traditional analysis by up to 10%. In addition, the variability of measurements at several HRs was reduced by up to 100 ms.

Novel T1 Analysis Method to Address Reduced Measurement Accuracy Due to Irregular Heart Rate Variability in Myocardial T1 Mapping Using Polarity-corrected Inversion Time Preparation.

PURPOSE: Polarity-corrected inversion time preparation (PCTIP), a myocardial T1 mapping technique, is expected to reduce measurement underestimation in the modified Look-Locker inversion recover method. However, measurement precision is reduced, especially for heart rate variability. We devised an analysis using a recurrence formula to overcome this problem and showed that it improved the measurement accuracy, especially at high heart rates. Therefore, this study aimed to determine the effect of this analysis on the accuracy and precision of T1 measurements for irregular heart rate variability. METHODS: A PCTIP scan using a 3T MRI scanner was performed in phantom experiment. We generated the simulated R-waves required for electrocardiogram (ECG)-gated acquisition using a signal generator set to 30 combinations. T1 map was generated using the signal train of the PCTIP images by nonlinear curve fitting using conventional and recurrence formulas. Accuracy against reference T1 and precision of heart rate variability were evaluated. To evaluate the fitting accuracy of both analyses, the relative fitting error was calculated. RESULTS: For the longer T1, the fitting error was larger than the short T1, with the conventional analysis showing 10.1±2.0%. The recurrence formula analysis showed a small fitting error less than 1%, which was consistent for all heart rate variability patterns. In the conventional analysis, the accuracy, especially for longer T1, showed a large underestimation of the measurements and poor linearity. However, in the recurrence formula analysis, the accuracy improved at a long T1, and linearity also improved. The Bland-Altman plot showed that it varied greatly depending on the heart rate variability pattern for the longer T1 in the conventional analysis, whereas the recurrence formula analysis suppressed this variation. CONCLUSION: T1 analysis of PCTIP using the recurrence formula analysis achieved accurate and precise T1 measurements, even for irregular heart rate variability.

SPON1 is an independent prognostic biomarker for ovarian cancer.

BACKGROUND: Ovarian cancer has the worst outcome among gynecological malignancies; therefore, biomarkers that could contribute to the early diagnosis and/or prognosis prediction are urgently required. In the present study, we focused on the secreted protein spondin-1 (SPON1) and clarified the prognostic relevance in ovarian cancer. METHODS: We developed a monoclonal antibody (mAb) that selectively recognizes SPON1. Using this specific mAb, we determined the expression of SPON1 protein in the normal ovary, serous tubal intraepithelial carcinoma (STIC), and ovarian cancer tissues, as well as in various normal adult tissues by immunohistochemistry, and verified its clinicopathological significance in ovarian cancer. RESULTS: The normal ovarian tissue was barely positive for SPON1, and no immunoreactive signals were detected in other healthy tissues examined, which was in good agreement with data obtained from gene expression databases. By contrast, upon semi-quantification, 22 of 242 ovarian cancer cases (9.1%) exhibited high SPON1 expression, whereas 64 (26.4%), 87 (36.0%), and 69 (28.5%) cases, which were designated as SPON1-low, possessed the moderate, weak, and negative SPON1 expression, respectively. The STIC tissues also possessed SPON1-positive signals. The 5-year recurrence-free survival (RFS) rate in the SPON1-high group (13.6%) was significantly lower than that in the SPON1-low group (51.2%). In addition, high SPON1 expression was significantly associated with several clinicopathological variables. Multivariable analysis revealed that high SPON1 was an independent prognostic factor for RFS of ovarian cancer. CONCLUSIONS: SPON1 represents a prognostic biomarker for ovarian cancer, and the anti-SPON1 mAb could be valuable as an outcome predictor.

A novel myocardial T1 analysis method robust to fluctuations in longitudinal magnetization recovery due to heart rate variability in polarity-corrected inversion time preparation.

Myocardial T1 mapping is useful for characterizing the myocardial tissues. Polarity-corrected inversion time preparation (PCTIP), one of the T1 mapping techniques, was expected to reduce measurement underestimation versus the MOLLI method. However, measurement accuracy is reportedly reduced, especially at high heart rates (HR), owing to the shorter time interval of inversion recovery (IR) pulses. This phantom-based experiment aimed to evaluate the dependence of T1 mapping with PCTIP on HR. Here we proposed and evaluated the effectiveness of a novel HR-independent analysis method for T1 mapping. A PCTIP scan using a 3-T magnetic resonance imaging scanner was performed on a T1 measurement phantom. The virtual HR were set at 50, 60, 75, and 100 bpm. The T1 of the phantom was estimated by a least-squares fit of the PCTIP data for each obtained inversion time and a theoretical longitudinal relaxation formula. This analysis was performed for the conventional and proposed formulas. The proposed formula was derived for adapting to the transient state of longitudinal magnetization recovery caused by the trigger interval as a recurrence formula. The estimated T1 measurements using the conventional formula varied widely with HR and the accuracy decreased, especially at a high HR. However, the proposed analysis showed good accuracy versus the conventional method independent of HR. T1 mapping using the PCTIP method combined with the novel method proposed here showed good accuracy.

Interplay between MPIase, YidC, and PMF during Sec-independent insertion of membrane proteins.

Integral membrane proteins with the N-out topology are inserted into membranes usually in YidC- and PMF-dependent manners. The molecular basis of the various dependencies on insertion factors is not fully understood. A model protein, Pf3-Lep, is inserted independently of both YidC and PMF, whereas the V15D mutant requires both YidC and PMF in vivo. We analyzed the mechanisms that determine the insertion factor dependency in vitro. Glycolipid MPIase was required for insertion of both proteins because MPIase depletion caused a significant defect in insertion. On the other hand, YidC depletion and PMF dissipation had no effects on Pf3-Lep insertion, whereas V15D insertion was reduced. We reconstituted (proteo)liposomes containing MPIase, YidC, and/or F0F1-ATPase. MPIase was essential for insertion of both proteins. YidC and PMF stimulated Pf3-Lep insertion as the synthesis level increased. V15D insertion was stimulated by both YidC and PMF irrespective of the synthesis level. These results indicate that charges in the N-terminal region and the synthesis level are the determinants of YidC and PMF dependencies with the interplay between MPIase, YidC, and PMF.

A rare case of recurrent ovarian cancer with TPM3-NTRK1 gene rearrangement: A case report.

NTRK gene fusion is rare in gynecological cancer. Entrectinib is a novel targeted drug, which is a potent inhibitor of TRK A, B and C. The present case report described a case of recurrent ovarian cancer with TPM3-NTRK1 rearrangement, which was detected by next-generation sequencing (NGS) and treated with entrectinib. A 56-year-old woman was diagnosed as having stage IV ovarian cancer with positive pleural fluid cytology. Neoadjuvant chemotherapy and interval debulking surgery, followed by chemotherapy, were performed. A total of 10 months after completion of chemotherapy, the disease recurred and the patient was treated with multimodal therapy for recurrence. DNA-based NGS detected TPM3-NTRK1 rearrangement and entrectinib therapy was initiated; however, the disease progressed despite 6 weeks of entrectinib administration, and 1 month after discontinuation of entrectinib, the patient died. After their death, immunohistochemistry with a pan-Trk monoclonal antibody was performed to determine the expression levels of TRK; however, immunohistochemistry was negative for TRK. In conclusion, the present case report described a rare case of recurrent ovarian cancer with TPM3-NTRK1 gene fusion, in which entrectinib was not effective. While NTRK gene fusion was detected by DNA-based NGS, immunohistochemistry was negative for TRK. These findings indicated that immunohistochemistry may be required for confirmation of TRK protein expression prior to entrectinib administration.

Successful management of pelvic recurrence of MSI-High endometrial cancer by total pelvic exenteration followed by administration of pembrolizumab:A case report.

Surgery can be curative treatment for pelvic locoregional recurrence of endometrial cancer; however, a cure is contingent on complete resection. Here, we report the case of a patient in whom recurrent endometrial tumor remained in the pelvis after resection; long-term control was achieved with postoperative administration of pembrolizumab.The patient had recurrent endometrial cancer of stage IA and was treated with chemotherapy and radiation, but tumor persisted in the pelvic cavity. We therefore attempted total pelvic exenteration, but the tumor was adherent to the pelvic wall and complete resection could not be achieved. However, postoperative administration of pembrolizumab controlled the residual tumor for more than two years without regrowth. We believe that since the resected tumor was MSI-High, the residual tumor responded well to pembrolizumab. It is not known whether cytoreductive surgery contributes to a long-term response to pembrolizumab, but at least in our patient, pembrolizumab appeared to be a very effective drug therapy for MSI-High endometrial cancer that was refractory to chemotherapy and radiotherapy.

Association of Chlamydia trachomatis infection with pregnancy outcomes among Japanese pregnant women: The Japan environment and children's study.

This study aimed to investigate the impact of Chlamydia trachomatis (CT) infection on pregnancy outcome in pregnant Japanese women. We utilized the data from a nationwide birth cohort study, the Japan Environment and Children's Study (JECS), for this study. We enrolled 26,385 individuals who could refer to data on pregnancy outcomes and confounding factors, with data on CT. Binominal logistic regression models were used to determine whether pregnant women with CT positivity were at more risk of experiencing adverse pregnancy outcomes, preterm birth (PTB), preterm prelabor rupture of membrane (pPROM), low birth weight (LBW) infants, small for gestational age (SGA) births, or hypertensive disorders of pregnancy (HDP). After adjusting for maternal age, parity, marital status, smoking status, and education status, there were no significantly increased risks of PTB, pPROM, LBW infants, SGA, and HDP in the odds ratios. No significant increase in the risk of adverse pregnancy outcomes was observed in any of the subgroup analyses, which were limited to the pregnancy women in Fukushima prefecture, where CT screening could be confirmed at 28-30 weeks of gestation. We believe that the results of this study will make a significant contribution to the future of medical care for pregnant women in Japan. Our findings are important for medical practitioners to contribute to the future medical treatment of Japanese pregnant women, and also to contribute to pre-conception care for Japanese society as a whole, including pregnant women.