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Primary cilia have specific proteins on their membrane to fulfill their sensory functions. Preservation of the specific protein composition of cilia relies on the barrier function of the transition zone (TZ) located at the ciliary base. Defects in cilia and the TZ cause ciliopathies, which have diverse clinical manifestations, including Meckel syndrome (MKS). Many of the proteins mutated in individuals with MKS are known to constitute the MKS module of the TZ. Although TXNDC15 (also known as MKS14) is a thioredoxin-related transmembrane protein that is localized mainly in the endoplasmic reticulum (ER) and is mutated in individuals with MKS, its role at the TZ or within cilia has not been characterized. Here, we show that TXNDC15-knockout cells have defects in MKS module assembly and in ciliary membrane protein localization. These defects in TXNDC15-knockout cells were not rescued by exogenous expression of any of the TXNDC15 constructs with MKS variations in the thioredoxin domain. Furthermore, TXNDC15 with mutations of two cysteine residues within the thioredoxin domain failed to rescue defects in TXNDC15-knockout cells, suggesting that TXNDC15 controls the TZ integrity from outside the TZ via its thioredoxin domain., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Background: No previous reports have characterized national profiles of soft-tissue sarcoma overall. We examined the nationwide statistics for soft-tissue sarcoma in Japan using data from the population-based National Cancer Registry., Methods: We identified 23 522 soft-tissue-sarcoma patients who were entered in the National Cancer Registry during 2016-19 using International Classification of Diseases-Oncology, Third Edition codes for cancer topography and morphology. We extracted data on patient demographics, tumor details (reason for diagnosis, tumor location, histology, extent of disease), hospital volume/type, treatment, and prognosis for each patient., Results: Soft-tissue sarcoma showed a slight male preponderance. Approximately 5500-6000 new cases were diagnosed as soft-tissue sarcoma per year, with the age-adjusted incidence of soft-tissue sarcoma being 3.22/100000/year. The age distribution showed a single peak in the 70-79 age range, and sex-stratified data showed it was higher in men. The most common histologic subtype was liposarcoma. The most frequent tumor locations were the soft tissue and skin, followed by the retroperitoneum. Extent of disease was categorized as: "localized" (31.3%), "regional" (38.9%), or "distant" (10.5%). We found significant associations between overall survival and sex, age, tumor location, facility type, hospital volume, reason for diagnosis, extent of disease, and surgical treatment., Conclusions: This is the first study to outline the epidemiology, clinical features, treatment, prognosis, and significant factors affecting prognosis of soft-tissue sarcoma in Japan using the National Cancer Registry. Documenting our data regarding elderly patients' outcomes is essential so other countries showing similar population-aging trends can learn from our experiences., Level of Evidence: Prognostic studies, Level III., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Although therapeutic human papillomavirus vaccines could offer a noninvasive treatment for patients with cervical intraepithelial neoplasia, none has been clinically implemented. Oral administration of the therapeutic human papillomavirus vaccine IGMKK16E7 results in the histological regression of human papillomavirus 16-positive cervical intraepithelial neoplasia 2/3 to normal (complete response). We investigated biomarkers that could predict complete response after oral administration of IGMKK16E7., Methods: Forty-two patients administered high-dose oral IGMKK16E7 in a phase I/II trial were included. Cervix-exfoliated cells were collected before vaccine administration. Gene expression of CD4, CD8, FOXP3, programmed cell death 1 protein, CTLA4, CD103, CD28, CD80, CD86, and programmed cell death 1 ligand 1 in the cells was measured by quantitative reverse transcriptase-polymerase chain reaction. Receiver operating characteristic curve analysis and Mann-Whitney tests were used to explore potential biomarkers. Pearson correlation coefficient analysis was used to correlate gene expression profiles with clinical outcome., Results: The only predictive biomarker of vaccine response for which receiver operating characteristic curve analysis showed significant diagnostic performance with histological complete response was CD86 (area under the curve = 0.71, 95% confidence interval = 0.53 to 0.88, P = .020). Patients with complete response had significantly lower CD86 expression (CD86-low) than patients with no complete response (P = .035). The complete response rates for CD86-low and CD86-high patients were 50% and 19%, respectively, and CD86-low patients had a significantly higher complete response rate (P = .047). Compared with all patients, the CD86-low group had a 1.5-fold increase in the complete response rate. Gene expression of CD86 and CTLA4 showed the strongest positive correlation with clinical outcomes in the incomplete response group (P < .001)., Conclusion: Low expression of CD86 in exfoliated cervical cells can be used as a pretreatment biomarker to predict histological complete response after IGMKK16E7 administration., (© The Author(s) 2024. Published by Oxford University Press.)

Junctional epidermolysis bullosa is an intractable cutaneous disorder in humans causing skin fragility and blistering due to mutations in genes encoding essential molecules adhering epidermis and dermis including collagen XVII. However, the pathogenesis still remains to be not fully understood perhaps because of a lack of appropriate animal models. In this study, we report novel mutant rats experiencing junctional epidermolysis bullosa, which was confirmed to be caused by a frameshift mutation of Col17a1 gene, as a rat model for investigating the underlying mechanism of pathogenesis. The mutant rats completely lacked the expression of collagen XVII and had blisters leading to infantile deaths as a homozygous condition, although their skin was apparently normal at birth by light microscopic evaluation except that immunohistochemical examination could not detect collagen XVII in any organs. These observations suggest that collagen XVII is not essential for the development of skin during the prenatal period but is indispensable for keeping epidermal-dermal connections stable after birth. Subsequent electron microscopic examinations further revealed an absence of hemidesmosomal inner plaques being composed of BP230, a binding partner of collagen XVII, and plectin in Col17a1-null newborns, albeit mRNA expressions of these molecules seemed to be unaffected at least during the fetal period. These results suggest that the lack of collagen XVII induces attenuation of hemidesmosomal inner plaques, which in turn destabilizes the epidermis-dermis connection and results in deterioration of epidermal physiology with formation of blisters after birth., (Copyright © 2024 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

Despite widespread cervical cancer (CC) screening programs, low participation has led to high morbidity and mortality rates, especially in developing countries. Because early-stage CC often has no symptoms, a non-invasive and convenient diagnostic method is needed to improve disease detection. In this study, we developed a new approach for differentiating both CC and cervical intraepithelial neoplasia (CIN)2/3, a precancerous lesion, from healthy individuals by exploring CC fatty acid metabolic reprogramming. Analysis of public datasets suggested that various fatty acid metabolizing enzymes were expressed at higher levels in CC tissues than in normal tissues. Correspondingly, 11 free fatty acids (FFAs) showed significantly different serum levels in CC patient samples compared with healthy donor samples. Nine of these 11 FFAs also displayed significant alterations in CIN2/3 patients. We then generated diagnostic models using combinations of these FFAs, with the optimal model including stearic and dihomo-γ-linolenic acids. Receiver operating characteristic curve analyses suggested that this diagnostic model could detect CC and CIN2/3 more accurately than using serum squamous cell carcinoma antigen level. In addition, the diagnostic model using FFAs was able to detect patients regardless of clinical stage or histological type. Overall, the serum FFA diagnostic model developed in this study could be a powerful new tool for the non-invasive early detection of CC and CIN2/3., (© 2024. The Author(s).)

Primary cilia are antenna-like structures protruding from the surface of various eukaryotic cells, and have distinct protein compositions in their membranes. This distinct protein composition is maintained by the presence of the transition zone (TZ) at the ciliary base, which acts as a diffusion barrier between the ciliary and plasma membranes. Defects in cilia and the TZ are known to cause a group of disorders collectively called the ciliopathies, which demonstrate a broad spectrum of clinical features, such as perinatally lethal Meckel syndrome (MKS), relatively mild Joubert syndrome (JBTS), and nonsyndromic nephronophthisis (NPHP). Proteins constituting the TZ can be grouped into the MKS and NPHP modules. The MKS module is composed of several transmembrane proteins and three soluble proteins. TMEM218 was recently reported to be mutated in individuals diagnosed as MKS and JBTS. However, little is known about how TMEM218 mutations found in MKS and JBTS affect the functions of cilia. In this study, we found that ciliary membrane proteins were not localized to cilia in TMEM218-knockout cells, indicating impaired barrier function of the TZ. Furthermore, the exogenous expression of JBTS-associated TMEM218 variants but not MKS-associated variants in TMEM218-knockout cells restored the localization of ciliary membrane proteins. In particular, when expressed in TMEM218-knockout cells, the TMEM218(R115H) variant found in JBTS was able to restore the barrier function of cells, whereas the MKS variant TMEM218(R115C) could not. Thus, the severity of symptoms of MKS and JBTS individuals appears to correlate with the degree of their ciliary defects at the cellular level., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Washing with water is not inferior to washing with soaps and detergents in children with atopic dermatitis (AD) in remission during the fall-winter seasons. We investigated whether this finding varies during summer based on the type of cleanser (soaps and detergents)., Methods: This evaluator-blinded, pragmatic, randomized, and non-inferiority study enrolled patients with AD whose eczema was controlled following regular steroid ointment application 2 days/week. For 8 ± 4 weeks, participants washed their upper and lower limbs with a cleanser on one side and with water alone on the other. Each participant chose either a weakly alkaline soap or an acidic detergent. The primary outcome was the Eczema Area and Severity Index (EASI) score at week 8 ± 4., Results: The data of 43 of the 47 registered participants were analyzed. The median patient age was 44 (23-99) months; 28 and 15 participants chose weakly alkaline and acidic cleansers, respectively. At week 8 ± 4, EASI scores of the water and cleanser sides were 0.00 (0.00-0.40) and 0.15 (0.00-0.40), respectively (p = 0.74). The difference between both sides was 0.00 (-0.07 to 0.14); the limits of the 95 % confidence interval did not reach the pre-specified non-inferiority margin. No difference was observed in the median Patient-Oriented Eczema Measure score, number of additional steroid ointment applications, and occurrences of skin infections. There were no differences between the cleanser types in any of the results., Conclusions: We demonstrated that washing with water was not inferior to that with a cleanser in patients with AD in the maintenance phase during summer, regardless of the type of cleanser., (Copyright © 2024 Japanese Society of Allergology. Published by Elsevier B.V. All rights reserved.)

Stearoyl-CoA desaturase 1 (SCD1) is an attractive target for cancer therapy. However, the clinical efficacy of SCD1 inhibitor monotherapy is limited. There is thus a need to elucidate the mechanisms of resistance to SCD1 inhibition and develop new therapeutic strategies for combination therapy. In this study, we investigated the molecular mechanisms by which cancer cells acquire resistance to endoplasmic reticulum (ER) stress-dependent cancer cell death induced by SCD1 inhibition. SCD1 inhibitor-sensitive and -resistant cancer cells were treated with SCD1 inhibitors in vitro, and SCD1 inhibitor-sensitive cancer cells accumulated palmitic acid and underwent ER stress response-induced cell death. Conversely, SCD1-resistant cancer cells did not undergo ER stress response-induced cell death because fatty acid desaturase 2 (FADS2) eliminated the accumulation of palmitic acid. Furthermore, genetic depletion using siRNA showed that FADS2 is a key determinant of sensitivity/resistance of cancer cells to SCD1 inhibitor. A549 cells, an SCD1 inhibitor-resistant cancer cell line, underwent ER stress-dependent cancer cell death upon dual inhibition of SCD1 and FADS2. Thus, combination therapy with SCD1 inhibition and FADS2 inhibition is potentially a new cancer therapeutic strategy targeting fatty acid metabolism., (© 2024. The Author(s).)

We previously reported that the inhibition of stearoyl-CoA desaturase 1 (SCD1) enhances the antitumor function of CD8 + T cells indirectly via restoring production of DC recruiting chemokines by cancer cells and subsequent induction of antitumor CD8 + T cells. In this study, we investigated the molecular mechanism of direct enhancing effects of SCD1 inhibitors on CD8 + T cells. In vitro treatment of CD8 + T cells with SCD1 inhibitors enhanced IFN-γ production and cytotoxic activity of T cells along with decreased oleic acid and esterified cholesterol, which is generated by cholesterol esterase, acetyl-CoA acetyltransferase 1 (ACAT1), in CD8 + T cells. The addition of oleic acid or cholesteryl oleate reversed the enhanced functions of CD8 + T cells treated with SCD1 inhibitors. Systemic administration of SCD1 inhibitor to MCA205 tumor-bearing mice enhanced IFN-γ production of tumor-infiltrating CD8 + T cells, in which oleic acid and esterified cholesterol, but not cholesterol, were decreased. These results indicated that SCD1 suppressed effector functions of CD8 + T cells through the increased esterified cholesterol in an ACAT1-dependent manner, and SCD1 inhibition enhanced T cell activity directly through decreased esterified cholesterol. Finally, SCD1 inhibitors or ACAT1 inhibitors synergistically enhanced the antitumor effects of anti-PD-1 antibody therapy or CAR-T cell therapy in mouse tumor models. Therefore, the SCD1-ACAT1 axis is regulating effector functions of CD8 + T cells, and SCD1 inhibitors, and ACAT1 inhibitors are attractive drugs for cancer immunotherapy., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Immunogenic responses by protein therapeutics often lead to reduced therapeutic effects and/or adverse effects via the generation of neutralizing antibodies and/or antidrug antibodies (ADA). Mirror-image proteins of the variable domain of the heavy chain of the heavy chain antibody (VHH) are potential novel protein therapeutics with high-affinity binding to target proteins and reduced immunogenicity because these mirror-image VHHs (d-VHHs) are less susceptible to proteolytic degradation in antigen-presenting cells (APCs). In this study, we investigated the preparation protocols of d-VHHs and their biological properties, including stereoselective target binding and immunogenicity. Initially, we established a facile synthetic process of two model VHHs [anti-GFP VHH and PMP12A2h1 (monomeric VHH of caplacizumab)] and their mirror-image proteins by three-step native chemical ligations (NCLs) from four peptide segments. The folded synthetic VHHs (l-anti-GFP VHH and l-PMP12A2h1) bound to the target proteins (EGFP and vWF-A1 domain, respectively), while their mirror-image proteins (d-anti-GFP VHH and d-PMP12A2h1) showed no binding to the native proteins. For biodistribution studies, l-VHH and d-VHH with single radioactive indium diethylenetriamine-pentaacid ( 111 In-DTPA) labeling at the C-terminus were designed and synthesized by the established protocol. The distribution profiles were essentially similar between l-VHH and d-VHH, in which the probes accumulated in the kidney within 15 min after intravenous administration in mice, because of the small molecular size of VHHs. Comparative assessment of the immunogenicity responses revealed that d-VHH-induced levels of ADA generation were significantly lower than those of native VHH, regardless of the peptide sequences and administration routes. The resulting scaffold investigated should be applicable in the design of d-VHHs with various C-terminal CDR3 sequences, which can be identified by screening using display technologies.

Background: Although many human papillomavirus (HPV)-targeted therapeutic vaccines have been examined for efficacy in clinical trials, none have been translated into clinical use. These previous agents were mostly administered by intramuscular or subcutaneous injection to induce systemic immunity. We investigated the safety and therapeutic efficacy of an HPV-16 E7-expressing lacticaseibacillus-based oral vaccine., Methods: In a double-blind, placebo-controlled, randomized trial, a total of 165 patients with HPV-16-positive high-grade cervical intraepithelial neoplasia 2 and 3 were assigned to orally administered placebo or low, intermediate, or high doses of IGMKK16E7 (lacticaseibacillus paracasei expressing cell surface, full-length HPV-16 E7). In the 4 groups, IGMKK16E7 or placebo was administered orally at weeks 1, 2, 4, and 8 postenrollment. The primary outcomes included histopathological regression and IGMKK16E7 safety., Results: In per-protocol analyses, histopathological regression to normal (complete response) occurred in 13 (31.7%) of 41 high-dose recipients and in 5 (12.5%) of 40 placebo recipients (rate difference = 19.2, 95% confidence interval [CI] = 0.5 to 37.8). In patients positive for HPV-16 only, the clinical response rate was 40.0% (12 of 30) in high-dose recipients and 11.5% (3 of 26) in recipients of placebo (rate difference = 28.5, 95% CI = 4.3 to 50.0). There was no difference in adverse events that occurred in the high-dose and placebo groups (P = .83). The number of HPV-16 E7-specific interferon-γ producing cells within peripheral blood increased with level of response (stable disease, partial, and complete responses; P = .004). The regression to normal (complete response) rates among recipients with high levels of immune response were increased in a dose-dependent manner., Conclusion: This trial demonstrates safety of IGMKK16E7 and its efficacy against HPV-16-positive cervical intraepithelial neoplasia 2 and 3. IGMKK16E7 is the first oral immunotherapeutic vaccine to show antineoplastic effects., Trial Registration: jRCT2031190034., (© The Author(s) 2023. Published by Oxford University Press.)

Lipopolysaccharide (LPS) is an endotoxin that can cause an acute inflammatory response. Nitric oxide (NO) is one of the most important innate immune system components and is synthesized by inducible NOS (iNOS) in macrophages in response to stimulation with LPS. LPS activates the RAS-RAF-mitogen-activated protein kinase/ERK kinase (MEK)-extracellular-signal-regulated kinase (ERK) signaling cascade in macrophages. The purpose of this study was to examine how the combination of LPS and MEK inhibitors, which have been used as anticancer agents in recent years, affects inflammation. We showed that MEK inhibitors enhanced iNOS expression and NO production in LPS-stimulated mouse bone marrow-derived macrophages. A MEK inhibitor increased the mortality rate in mice with LPS-induced inflammation. The expression of the cytokine interleukin-12 (IL-12) in macrophages was enhanced by the MEK inhibitor, as shown by a cytokine array and ELISA. IL-12 enhanced iNOS expression and NO production in response to LPS. We also showed that tumor necrosis factor (TNF-α) was secreted by macrophage after stimulation with LPS and that TNF-α and IL-12 synergistically induced iNOS expression and NO production. An anti-IL-12 neutralizing antibody prevented NO production and mortality in an LPS-induced inflammation mouse model in the presence of a MEK inhibitor. These results suggest that the MEK inhibitor increases the mortality rate in mice with LPS-induced inflammation through IL-12-NO signaling., (© 2023. Cell Death Differentiation Association (ADMC).)

Owing to their crucial roles in development and homeostasis, defects in cilia cause ciliopathies with diverse clinical manifestations. The intraflagellar transport (IFT) machinery, containing the IFT-A and IFT-B complexes, mediates not only the intraciliary bidirectional trafficking but also import and export of ciliary proteins together with the kinesin-2 and dynein-2 motor complexes. The BBSome, containing eight subunits encoded by causative genes of Bardet-Biedl syndrome (BBS), connects the IFT machinery to ciliary membrane proteins to mediate their export from cilia. Although mutations in subunits of the IFT-A and dynein-2 complexes cause skeletal ciliopathies, mutations in some IFT-B subunits are also known to cause skeletal ciliopathies. We here show that compound heterozygous variations of an IFT-B subunit, IFT81, found in a patient with skeletal ciliopathy cause defects in its interactions with other IFT-B subunits, and in ciliogenesis and ciliary protein trafficking when one of the two variants was expressed in IFT81-knockout (KO) cells. Notably, we found that IFT81-KO cells expressing IFT81(Δ490-519), which lacks the binding site for the IFT25-IFT27 dimer, causes ciliary defects reminiscent of those found in BBS cells and those in IFT74-KO cells expressing a BBS variant of IFT74, which forms a heterodimer with IFT81. In addition, IFT81-KO cells expressing IFT81(Δ490-519) in combination with the other variant, IFT81 (L645*), which mimics the cellular conditions of the above skeletal ciliopathy patient, demonstrated essentially the same phenotype as those expressing only IFT81(Δ490-519). Thus, our data indicate that BBS-like defects can be caused by skeletal ciliopathy variants of IFT81., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Ovarian cancer has a poor prognosis and is difficult to detect in early stages. Therefore, developing new diagnostic markers for early-stage ovarian cancer is critical. Here, we developed a diagnostic marker for early-stage ovarian cancer on the basis of fatty acid metabolism characteristics of cancer cells. The expression of various fatty acid metabolizing enzymes such as stearoyl-CoA desaturase 1 (SCD1) was altered in early-stage ovarian cancer tissue compared with that in normal ovarian tissue. Changes in the expression of fatty acid metabolizing enzymes, particularly SCD1, in cancer tissues were found to alter concentrations of multiple free fatty acids (FFA) in serum. We were the first to show that fatty acid metabolic characteristics in tissues are related to the FFA composition of serum. Surprisingly, patients with stage I/II ovarian cancer also showed significant changes in serum levels of eight FFAs, which can be early diagnostic markers. Finally, using statistical analysis, an optimal early diagnostic model combining oleic and arachidic acid levels, fatty acids associated with SCD1, was established and confirmed to have higher diagnostic power than CA125, regardless of histology. Thus, our newly developed diagnostic model using serum FFAs may be a powerful tool for the noninvasive early detection of ovarian cancer., Significance: Measurement of serum FFA levels by changes in the expression of fatty acid metabolizing enzymes in tumor tissue would allow early detection of ovarian cancer. In particular, the SCD1-associated FFAs, oleic and arachidic acid, would be powerful new screening tools for early-stage ovarian cancer., (© 2023 The Authors; Published by the American Association for Cancer Research.)

A 78-year-old Japanese male with previous gastric cancer and untreated diabetes mellitus was admitted to hospital for persistent fever and leg edema. Blood culture was positive for Streptococcus angino'sus, and echocardiography showed isolated tricuspid valve infective endocarditis. Infection was controlled with intravenous antibiotics, but surgery was indicated because of persistent severe regurgitation and large vegetation of 15 mm in size. As the tricuspid valve anterior leaflet was extensively damaged, he underwent valve replacement using a bioprosthetic valve. The patient was discharged 25 days postoperatively with additional antibiotics, and he has been free from recurrent endocarditis for 6 months.

Within cilia, the dynein-2 complex needs to be transported as an anterograde cargo to achieve its role as a motor to drive retrograde trafficking of the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes. We previously showed that interactions of WDR60 and the DYNC2H1-DYNC2LI1 dimer of dynein-2 with multiple IFT-B subunits, including IFT54, are required for the trafficking of dynein-2 as an IFT cargo. However, specific deletion of the IFT54-binding site from WDR60 demonstrated only a minor effect on dynein-2 trafficking and function. We here show that the C-terminal coiled-coil region of IFT54, which participates in its interaction with the DYNC2H1-DYNC2LI1 dimer of dynein-2 and with IFT20 of the IFT-B complex, is essential for IFT-B function, and suggest that the IFT54 middle linker region between the N-terminal WDR60-binding region and the C-terminal coiled-coil is required for ciliary retrograde trafficking, probably by mediating the effective binding of IFT-B to the dynein-2 complex, and thereby ensuring dynein-2 loading onto the anterograde IFT trains. The results presented here agree with the notion predicted from the previous structural models that the dynein-2 loading onto the anterograde IFT train relies on intricate, multivalent interactions between the dynein-2 and IFT-B complexes., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The primary cilium is a sensory organelle, receiving signals from the external environment and relaying them into the cell. Mutations in proteins required for transport in the primary cilium result in ciliopathies, a group of genetic disorders that commonly lead to the malformation of organs such as the kidney, liver and eyes and skeletal dysplasias. The motor proteins dynein-2 and kinesin-2 mediate retrograde and anterograde transport, respectively, in the cilium. WDR34 (also known as DYNC2I2), a dynein-2 intermediate chain, is required for the maintenance of cilia function. Here, we investigated WDR34 mutations identified in Jeune syndrome, short-rib polydactyly syndrome and asphyxiating thoracic dysplasia patients. There is a poor correlation between genotype and phenotype in these cases, making diagnosis and treatment highly complex. We set out to define the biological impacts on cilia formation and function of WDR34 mutations by stably expressing the mutant proteins in WDR34-knockout cells. WDR34 mutations led to different spectrums of phenotypes. Quantitative proteomics demonstrated changes in dynein-2 assembly, whereas initiation and extension of the axoneme, localization of intraflagellar transport complex-B proteins, transition zone integrity and Hedgehog signalling were also affected., Competing Interests: Competing interests D.J.S. is an Editor for Journal of Cell Science and played no role in the editorial handling of this paper. The authors declare no other competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The dynein-2 complex must be transported anterogradely within cilia to then drive retrograde trafficking of the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes. Here, we screened for potential interactions between the dynein-2 and IFT-B complexes and found multiple interactions among the dynein-2 and IFT-B subunits. In particular, WDR60 (also known as DYNC2I1) and the DYNC2H1-DYNC2LI1 dimer from dynein-2, and IFT54 (also known as TRAF3IP1) and IFT57 from IFT-B contribute to the dynein-2-IFT-B interactions. WDR60 interacts with IFT54 via a conserved region N-terminal to its light chain-binding regions. Expression of the WDR60 constructs in WDR60-knockout (KO) cells revealed that N-terminal truncation mutants lacking the IFT54-binding site fail to rescue abnormal phenotypes of WDR60-KO cells, such as aberrant accumulation of the IFT machinery around the ciliary tip and on the distal side of the transition zone. However, a WDR60 construct specifically lacking just the IFT54-binding site substantially restored the ciliary defects. In line with the current docking model of dynein-2 with the anterograde IFT trains, these results indicate that extensive interactions involving multiple subunits from the dynein-2 and IFT-B complexes participate in their connection., Competing Interests: Competing interests D.J.S. is an Editor for Journal of Cell Science and played no role in the editorial handling of this paper. The authors declare no other competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

A 72-year-old man with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) was treated with dasatinib (week1: 50 mg/day, week2: 70 mg/day, week3-: 100 mg/day) and prednisolone from June 2017. However, in January 2018, it relapsed with the T315I mutation. Although the treatment was changed to ponatinib 30 mg/day, he experienced a second relapse in June 2018. Following confirmation of CD22 positivity, he was treated with three cycles of inotuzumab ozogamicin (InO), resulting in CR. He was CR for 2.9 years before relapsing for the third time in May 2021. Because the patient was still CD22-positive, InO was given again, and the patient achieved CR at the end of the second cycle. We had a case where re-administering InO was effective as a salvage therapy for relapsed/refractory Ph+ALL (r/r Ph+ALL) in an elderly patient.

Atherosclerotic plaques develop from the accumulation of macrophage-derived foam cells via the uptake of modified low-density lipoprotein (LDL). CD36 and CD204 are the principal scavenger receptors responsible for the uptake of modified LDL. Although glucocorticoids are suspected to exacerbate atherosclerosis, the precise mechanisms have not been fully elucidated. We investigated the effects of long-term treatment (2 weeks) with both a natural glucocorticoid (hydrocortisone, HC, 1 μM) and a synthetic glucocorticoid (dexamethasone, Dex, 100 nM) on murine bone marrow-derived macrophages using flow cytometry and western blotting. Treatment with HC and Dex enhanced CD204 expression but not CD36 expression and acetylated LDL (Ac-LDL) uptake. Treatment with HC and Dex also induced the phosphorylation of extracellular signal-regulated kinase (ERK). The Dex-induced enhancement in CD204 expression and Ac-LDL uptake were suppressed by an inhibitor of the mitogen-activated protein kinase (MAPK)/ERK kinase. These results suggest that glucocorticoids activate the MAPK/ERK pathway, which enhances CD204 expression and results in increased uptake of Ac-LDL in macrophages. The MAPK/ERK pathway in macrophages might be a key target to prevent atherosclerosis that is worsened by glucocorticoids., Competing Interests: Declaration of competing interest None declared., (Copyright © 2022 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Objective: To identify candidate predictors for the prognosis of cervical intraepithelial neoplasia 2 (CIN2) lesions and evaluate the prognostic value of the local immune response., Methods: One hundred fifteen CIN2 patients were enrolled. The percentage of p16-, minichromosome maintenance complex component 2- or apolipoprotein B mRNA editing enzyme catalytic subunit 3G (APOBEC3G)-positive cells was determined immunohistochemically. Tumor-infiltrating lymphocytes (TILs) in intertumoral lesions were scored using an automated system. CIN3 disease progression and regression rates were estimated by the Kaplan-Meier method. A case-control study was conducted to screen CIN2 prognostic factors in 10 regression and 10 progression patients. Selected factors were examined in a cohort study to determine their prognostic value for CIN2., Results: Among all participants, the cumulative progression and regression rates at 60 months were 0.477 and 0.510, respectively. In the case-control study, p16- and APOBEC3G-positive cells were higher in the progression group (p=0.043, p=0.023). Additionally, CD4 + cell infiltration was enhanced in the regression group (p=0.023). The cohort study revealed a significantly increased progression rate in patients with elevated p16-positive cells (p<0.001), and increased CD4 + TIL infiltration was associated with better regression (p=0.011). Kaplan-Meier analysis according to human papillomavirus (HPV) positivity revealed a greater CIN3 development risk in HPV16-positive patients than in HPV16-negative cases. Finally, multivariate analysis identified HPV16 infection and CD4 + TIL infiltration as independent prognostic factors in CIN2 regression., Conclusion: CD4 + TIL infiltration in intertumoral lesions was related with CIN2 regression. Our findings suggest CD4 + TIL infiltration may be useful for the triage of CIN2 patients., Competing Interests: The authors declare no potential conflicts of interest., (© 2023. Asian Society of Gynecologic Oncology, Korean Society of Gynecologic Oncology, and Japan Society of Gynecologic Oncology.)

Preeclampsia (PE) is a hypertensive obstetric disorder with poor prognosis for both the mother and offspring. Infants born to mothers with PE are known to be at increased risk of developing higher brain dysfunction, such as autism. However, how maternal PE can affect the environment in the fetal brain has not been fully elucidated. Here, we examined the impact of PE on the fetal brain in a mouse model of PE induced by angiotensin II (Ang II), focusing on changes in the inflammatory condition. We confirmed that pregnant mice which were continuously administered Ang II exhibited PE phenotypes, including high blood pressure, proteinuria, and fetal growth restriction. Quantitative RT-PCR analysis demonstrated that the brain of fetuses on embryonic day 17.5 (E17.5) in the Ang II-administered pregnant mice showed increased expression of cytokines, interleukin (IL)- 6, IL-17a, tumor necrosis factor-α, interferon-γ, IL-12, IL-4, and IL-10. Immunohistochemical analysis over a wide area, from the tip of the frontal lobe to the posterior cerebral end, on E17.5 revealed that the microglia in the fetal brain of the Ang II-administered group displayed higher solidity and circularity than those of the control group, indicating that the microglia had transformed to an amoeboid morphology and were activated. Our findings suggest that maternal PE may cause altered inflammatory conditions in the fetal brain, which might be associated with the pathological mechanism connecting maternal PE and brain dysfunction in the offspring., Competing Interests: Conflicts of Interest The authors declare no conflicts of interest associated with this manuscript., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The intraflagellar transport (IFT) machinery mediates the import and export of ciliary proteins across the ciliary gate, as well as bidirectional protein trafficking within cilia. In addition to ciliary anterograde protein trafficking, the IFT-B complex participates in the export of membrane proteins together with the BBSome, which consists of eight subunits encoded by the causative genes of Bardet-Biedl syndrome (BBS). The IFT25-IFT27/BBS19 dimer in the IFT-B complex constitutes its interface with the BBSome. We show here that IFT25-IFT27 and the RABL2 GTPase bind the IFT74/BBS22-IFT81 dimer of the IFT-B complex in a mutually exclusive manner. Cells expressing GTP-locked RABL2 [RABL2(Q80L)], but not wild-type RABL2, phenocopied IFT27 -knockout cells, that is, they demonstrated BBS-associated ciliary defects, including accumulation of LZTFL1/BBS17 and the BBSome within cilia and the suppression of export of the ciliary GPCRs GPR161 and Smoothened. RABL2(Q80L) enters cilia in a manner dependent on the basal body protein CEP19, but its entry into cilia is not necessary for causing BBS-associated ciliary defects. These observations suggest that GTP-bound RABL2 is likely to be required for recruitment of the IFT-B complex to the ciliary base, where it is replaced with IFT25-IFT27.

Early diagnosis of malignant melanoma is critical for effective treatment and reduced patient mortality. However, current clinical and histological variables show limited accuracy in diagnosis. Serum or urine level of 5-S-cysteinyldopa (5-S-CD) is a commonly used melanoma biomarker in Japan owing to its increased sensitivity compared with other melanoma markers. However, its use as a diagnostic marker has shown some limitations. Therefore, here we examined the combination of 5-S-CD with melanoma inhibitory activity, which showed sensitivity in detecting melanoma comparable with that of 5-S-CD, and interleukin-8, a cytokine linked with melanoma progression, in a cohort of Japanese patients with melanoma. Our results revealed that the triple combination of 5-S-CD, melanoma inhibitory activity, and interleukin-8 showed high diagnostic accuracy in detecting melanoma compared with each of the individual factors. Importantly, the triple marker showed specificity and utility in detecting early-stage melanoma. Our results suggest the utility of the triple marker as a diagnostic biomarker for melanoma patients., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc.)

Bidirectional protein trafficking within cilia is mediated by the intraflagellar transport (IFT) machinery, which contains the IFT-A and IFT-B complexes powered by the kinesin-2 and dynein-2 motors. Mutations in genes encoding subunits of the IFT-A and dynein-2 complexes cause skeletal ciliopathies. Some subunits of the IFT-B complex, including IFT52, IFT80, and IFT172, are also mutated in skeletal ciliopathies. We here show that IFT52 variants found in individuals with short-rib polydactyly syndrome (SRPS) are compromised in terms of formation of the IFT-B holocomplex from two subcomplexes and its interaction with heterotrimeric kinesin-II. IFT52 -knockout (KO) cells expressing IFT52 variants that mimic the cellular conditions of individuals with SRPS demonstrated mild ciliogenesis defects and a decrease in ciliary IFT-B level. Furthermore, in IFT52 -KO cells expressing an SRPS variant of IFT52, ciliary tip localization of ICK/CILK1 and KIF17, both of which are likely to be transported to the tip via binding to the IFT-B complex, was significantly impaired. Altogether these results indicate that impaired anterograde trafficking caused by a decrease in the ciliary level of IFT-B or in its binding to kinesin-II underlies the ciliary defects found in skeletal ciliopathies caused by IFT52 variations.

Primary cilia are antenna-like organelles that contain specific proteins, and are crucial for tissue morphogenesis. Anterograde and retrograde trafficking of ciliary proteins are mediated by the intraflagellar transport (IFT) machinery. BROMI/TBC1D32 interacts with CCRK/CDK20, which phosphorylates and activates the intestinal cell kinase (ICK)/CILK1 kinase, to regulate the change in direction of the IFT machinery at the ciliary tip. Mutations in BROMI , CCRK , and ICK in humans cause ciliopathies, and mice defective in these genes are also known to demonstrate ciliopathy phenotypes. We show here that BROMI interacts not only with CCRK but also with CFAP20, an evolutionarily conserved ciliary protein, and with FAM149B1/ Joubert syndrome (JBTS)36, a protein in which mutations cause JBTS. In addition, we show that FAM149B1 interacts directly with CCRK as well as with BROMI. Ciliary defects observed in CCRK -knockout (KO), BROMI -KO, and FAM149B1 -KO cells, including abnormally long cilia and accumulation of the IFT machinery and ICK at the ciliary tip, resembled one another, and BROMI mutants that are defective in binding to CCRK and CFAP20 were unable to rescue the ciliary defects of BROMI -KO cells. These data indicate that CCRK, BROMI, FAM149B1, and probably CFAP20 altogether regulate the IFT turnaround process under the control of ICK.

Background: There is a paucity of data on predictors of clinical history in oral food challenge (OFC) outcome for the initial diagnosis of food protein-induced enterocolitis syndrome (FPIES)., Objective: This study aimed to identify predictors for the diagnosis of FPIES., Methods: The study included patients who underwent OFC to diagnose FPIES from 2010 to 2021. Patients with a positive OFC result were classified as belonging to the FPIES group, and those with negative OFC result within 120 days from the last symptomatic episode were classified as belonging to the no-allergy (NA) group. Background factors were analyzed in the groups., Results: A total of 50 OFCs to 12 different foods were conducted in 50 patients. Of those 50 patients, 30 were classified as belonging to the FPIES group. No significant difference was observed between the FPIES and NA groups with respect to background factors, including the features of symptomatic episodes and examinations of immediate-type allergy. A history of asymptomatic ingestion was observed in 23 of 24 and 13 of 19 patients in the FPIES and NA groups, respectively; thus, it was significantly more common in patients with FPIES. The diagnostic rate of patients with fewer than 3 symptomatic episodes was 52%, and that of patients with 3 episodes or more was 75%, not considering a patient without available data., Conclusions: A definite diagnosis of FPIES should be based on OFC, as there are no predictors for OFC positivity other than a history of asymptomatic ingestion. The absence of asymptomatic ingestion history was a negative predictor for the diagnosis of FPIES., (© 2022 The Author(s).)

Background: Understanding the mechanisms of non-T cell inflamed tumor microenvironment (TME) and their modulation are important to improve cancer immunotherapies such as immune checkpoint inhibitors. The involvement of various immunometabolisms has recently been indicated in the formation of immunosuppressive TME. In this study, we investigated the immunological roles of stearoyl-CoA desaturase 1 (SCD1), which is essential for fatty acid metabolism, in the cancer immune response., Methods: We investigated the roles of SCD1 by inhibition with the chemical inhibitor or genetic manipulation in antitumor T cell responses and the therapeutic effect of anti-programmed cell death protein 1 (anti-PD-1) antibody using various mouse tumor models, and their cellular and molecular mechanisms. The roles of SCD1 in human cancers were also investigated by gene expression analyses of colon cancer tissues and by evaluating the related free fatty acids in sera obtained from patients with non-small cell lung cancer who were treated with anti-PD-1 antibody., Results: Systemic administration of a SCD1 inhibitor in mouse tumor models enhanced production of CCL4 by cancer cells through reduction of Wnt/β-catenin signaling and by CD8 + effector T cells through reduction of endoplasmic reticulum stress. It in turn promoted recruitment of dendritic cells (DCs) into the tumors and enhanced the subsequent induction and tumor accumulation of antitumor CD8 + T cells. SCD1 inhibitor was also found to directly stimulate DCs and CD8 + T cells. Administration of SCD1 inhibitor or SCD1 knockout in mice synergized with an anti-PD-1 antibody for its antitumor effects in mouse tumor models. High SCD1 expression was observed in one of the non-T cell-inflamed subtypes in human colon cancer, and serum SCD1 related fatty acids were correlated with response rates and prognosis of patients with non-small lung cancer following anti-PD-1 antibody treatment., Conclusions: SCD1 expressed in cancer cells and immune cells causes immunoresistant conditions, and its inhibition augments antitumor T cells and therapeutic effects of anti-PD-1 antibody. Therefore, SCD1 is an attractive target for the development of new diagnostic and therapeutic strategies to improve current cancer immunotherapies including immune checkpoint inhibitors., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

The development of in vitro toxicity assessment methods using cultured cells has gained popularity for promoting animal welfare in animal experiments. Herein, we briefly discuss the current status of hepatoxicity assessment using human- and rat-derived hepatocytes; we focus on the liver organoid method, which has been extensively studied in recent years, and discuss how toxicologic pathologists can use their knowledge and experience to contribute to the development of in vitro chemical hepatotoxicity assessment methods for drugs, pesticides, and chemicals. We also propose how toxicological pathologists should assess toxicity regarding the putative distribution of undifferentiated and differentiated cells in the organoid when liver organoids are observed in hematoxylin and eosin-stained specimens. This was done while considering the usefulness and limitations of in vitro studies for toxicologic pathology assessment., (©2022 The Japanese Society of Toxicologic Pathology.)

The IFT-B complex mediates ciliary anterograde protein trafficking and membrane protein export together with the BBSome. Bardet-Biedl syndrome (BBS) is caused by mutations in not only all BBSome subunits but also in some IFT-B subunits, including IFT74/BBS22 and IFT27/BBS19, which form heterodimers with IFT81 and IFT25, respectively. We found that the IFT25-IFT27 dimer binds the C-terminal region of the IFT74-IFT81 dimer and that the IFT25-IFT27-binding region encompasses the region deleted in the BBS variants of IFT74. In addition, we found that the missense BBS variants of IFT27 are impaired in IFT74-IFT81 binding and are unable to rescue the BBS-like phenotypes of IFT27-knockout (KO) cells. Furthermore, the BBS variants of IFT74 rescued the ciliogenesis defect of IFT74-KO cells, but the rescued cells demonstrated BBS-like abnormal phenotypes. Taken together, we conclude that the impaired interaction between IFT74-IFT81 and IFT25-IFT27 causes the BBS-associated ciliary defects., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)