FOXM1 and CHD4 expression is associated with chemoresistance in hepatoblastoma.

Hepatoblastoma (HB) is the most common malignant liver tumor in childhood. Although pre-operative cisplatin (CDDP)-based chemotherapy is often used in cases of HB, about 20％ of HB patients exhibit resistance to CDDP. Forkhead box protein M1 (FOXM1) and chromo-domain-helicase-DNA-binding protein 4 (CHD4) have been associated with CDDP resistance in various tumors. We here analyzed the immunohistochemical expression of FOXM1 and CHD4 in HB specimens of 33 patients (mean age: 20 months) post-chemotherapy. The differentiation of specimens was assessed using the digital pathology software QuPath®, and then the relation between the FOXM1 or CHD4 expression and the differentiation and various other clinicopathological parameters was investigated. The histological type was epithelial in 19 cases (57.6%) and mixed epithelial and mesenchymal in 14 cases (42.4%). Nine cases had only a fetal component, 1 case had only an embryonal component, 22 cases had both fetal and embryonal components, and 1 case had no viable tumor. Both the FOXM1 and CHD4 immunoexpressions were found significantly more frequently in the embryonal than fetal components (p<0.0001 and p<0.0001, respectively). Regarding chemotherapy efficacy, the alpha-fetoprotein (AFP) level after chemotherapy was correlated with both the imaging shrinkage rate (R=-0.52) and histological residual rate (the percentage of the viable tumors of HB after chemotherapy)(R=0.62). High FOXM1 score was correlated with a high-postoperative AFP value (p<0.01) and a low AFP attenuation rate (p<0.05), but the FOXM1 score was not correlated with the imaging shrinkage rate (p=0.4418) or histological residual rate (p=0.4418). High CHD4 score showed a nonsignificant trend toward correlation with high postoperative AFP value (p=0.0849) and was not significantly correlated with the other parameters. Collectively, our results showed that FOXM1 expression may be useful in evaluating the response to CDDP-based chemotherapeutic regimens. Accurate measurement of FOXM1 expression by our scoring system using QuPath® is important in cases with mixed HB components of various differentiation levels.

Long-term cosmetic outcomes of the slit-slide procedure for umbilical hernia repair in children.

PURPOSE: To assess the long-term cosmetic outcomes of the "slit-slide procedure", designed to provide a more natural appearance for umbilical hernia repair, as perceived by the patients and their parents. METHODS: A total of 149 patients with umbilical hernia underwent the slit-slide procedure at our hospital. The slit-slide procedure allows for the creation of an umbilicus with a more natural and integrated appearance. The patient satisfaction survey questionnaire was mailed to the families (n = 139), and there were 74 (53.2%) respondents. A questionnaire survey on postoperative appearance was also distributed to pediatric surgeons. RESULTS: The median age at the time of operation was 2.5 years (range, 2 months to 14 years) and the average median age at the time of answering the questionnaire was 6.25 years old (range, 2.5-14.8 years). The average median period of observation was 3.2 years (range, 4 months to 8.97 years). Most patients (89.2%) and parents (93.2%) were satisfied with the appearance of the umbilicus. Twenty-seven patients reported improved satisfaction after surgery (36.2%). Surgeons tended to score the elongated-oval shape highly; however, there was no difference in satisfaction among the shapes. CONCLUSION: The slit-slide procedure is not only effective and safe, but it achieves a satisfactory aesthetic outcome.

Thoracoscopic repair for late-presenting congenital diaphragmatic hernia with thoracic kidney in a child.

Congenital diaphragmatic hernia (CDH) with a hernia sac and thoracic kidney is a very rare congenital anomaly. Recently, the usefulness of endoscopic surgery for CDH has been reported. We herein report a patient who underwent thoracoscopic repair of CDH with a hernia sac and thoracic kidney. A 7-year-old boy was referred to our hospital due to a diagnosis of CDH without clinical symptoms. Computed tomography showed herniation of the intestine into the left thorax and left-sided thoracic kidney. The key points of operation are resection of the hernia sac and identification of the suturable diaphragm under the presence of the thoracic kidney. In the present case, after repositioning the kidney to the subdiaphragmatic area completely, the border of the diaphragmatic rim was clearly visualized. Good visibility allowed resection of the hernia sac without damaging the phrenic nerve and closure of the diaphragmatic defect.

Feasibility of Laparoscopic Fundoplication Without Removing the Preceding Gastrostomy in Severely Neurologically Impaired Patients: A Multicenter Evaluation of the Traction Technique.

Purpose: Severely neurologically impaired patients sometimes require anti-reflux surgery with preceding gastrostomy. We apply a traction technique for laparoscopic fundoplication (LF) without gastrostomy takedown (GTD) in such cases. We conducted a multicenter review to assess the feasibility of our approach. Materials and Methods: In brief, the traction technique involves left-lateral-traction of the stomach body, right-lateral-traction of the round ligament of the liver, and elevation of the left liver lobe to create a sufficient field for manipulating the forceps. Patients who underwent LF with Nissen's procedures in 2010-2022 were retrospectively reviewed. Data were analyzed by a one-way analysis of variance. Results: The operative approaches included the traction technique (n = 16; Group 1), GTD and reconstruction (n = 5; Group 2), and LF followed by gastrostomy (n = 92; Group 3). In comparison with Group 1, significant differences were only found in pneumoperitoneum time (Group 1 versus Group 2 versus Group 3: 174.4 minutes versus 250.4 minutes versus 179.5 minutes; P = .0179). Operating time (222.7 minutes versus 303.0 minutes versus 239.7 minutes; P = .0743), duration to full-strength enteral nutrition (10.4 days versus 17.2 days versus 11.0 days; P = .0806), and length of hospital stay (17.2 days versus 31.0 days versus 18.5 days; P = .3247) were equivalent. No re-fundoplication was required in Group 1. Conclusion: The traction technique secures the operative quality and outcome of LF without GTD.

Spontaneous reduction age for ovarian hernia in early infancy.

BACKGROUND: Many articles recommend early surgery for ovarian hernia to avoid the risk of ovarian torsion. However, while ovarian hernia is known to undergo spontaneous reduction (SR) in early infancy, few reports have described the timing of SR. We therefore investigated the clinical features of SR for ovarian hernia in early infancy. METHODS: A total of 610 girls were diagnosed with inguinal hernia between 2008 and 2018. We focused on infants who had an ovarian hernia onset in the first 3 months of age. We reviewed the age retrospectively at the onset of hernia and age at SR. The data were compared statistically using the Kaplan-Meier method. RESULTS: Sixty-one infants with inguinal hernia were included in this study. Thirty-nine patients (64%) had ovarian hernia. The mean age at the onset of hernia was 44 ± 17 days of age. Thirty cases underwent SR (77%). A Kaplan-Meier analysis showed that 75% of ovarian hernias underwent SR by 6 months of age. There were no cases of ovarian torsion. CONCLUSIONS: Most cases of ovarian hernia underwent SR, so patients with ovarian hernia in early infancy might be treated by elective surgery after 6 months of age.

Support for Introduction of Pediatric Endosurgery in Nepal as Global Pediatric Surgery: Preliminary Needs Assessment Survey.

Background: Endosurgery provides several advantages over open surgery in the context of global surgery; however, there are several barriers to its introduction. The preliminary assessment of needs and barriers is essential for carrying out effective support as Global Surgery. However, no report has described the initiation of support for endosurgery based on a preliminary survey of the needs and barriers. The present survey study aimed at determining the needs and barriers of pediatric endosurgery in Nepal. Materials and Methods: A needs assessment survey was conducted among all pediatric surgeons in Nepal via an online platform. This was followed by workshop on pediatric endosurgical skills in Nepal. To assess the skills of participants and effectiveness of the workshop, the skill evaluation tests and the questionnaire survey were conducted. Results: Fourteen pediatric surgeons (response rate: 60.9%) responded to the needs assessment survey. More than 70% of the participants did not have any experience with advanced endosurgical procedures. However, advanced endosurgical procedures were strongly needed. A lack of training was indicated as a major barrier for the introduction of pediatric endosurgery. Fifteen participants completed the workshop. Participants' confidence in their endosurgical skills improved significantly after the workshop. The skill evaluation tests revealed that participants' endosurgical skills also improved significantly after the workshop, although even after the workshop, participants still took an average of 415.6 seconds to place and knot one suture. Conclusions: The needs assessment survey and workshop for Nepalese pediatric surgeons helped clarify their needs for endosurgery and the barriers to its introduction.

A totally extraperitoneal approach for endoscopic resection of a congenital prepubic sinus through the pubic symphysis.

BACKGROUND: A congenital prepubic sinus (CPS) is a rare congenital anomaly in which a duct remnant extends from the skin opening near the pubic symphysis to various parts and the lesions are mostly located in the preperitoneal space. The totally extraperitoneal (TEP) approach is an operational method that provides a good field of view for the preperitoneal space. We report the CPS through the pubic symphysis in which complete resection was achieved by a TEP approach. TEP approach was minimally invasive and achieved satisfactory cosmetic outcome. CASE PRESENTATION: We herein report the case of a 13-year-old boy with a fistula opening near the dorsal penis. He was admitted to our hospital due to fever and lower abdominal pain. Abdominal ultrasonography and computed tomography revealed an abscess inside a fistula lumen on the posterior surface of the rectus abdominis muscles in the midline of the lower abdomen. Under a diagnosis of CPS, which was located in the preperitoneal space, endoscopic resection was performed by a totally extraperitoneal approach. After making an umbilical incision, the rectus abdominis muscle was excised outward to expose the preperitoneal space. A single-port system was placed in the preperitoneal space. Three 5-mm-port trocars were inserted. As the preperitoneal cavity was expanded, a sinus connecting to the pubic symphysis was confirmed. The pubic symphysis did not connect with the bladder. Because the fistula was penetrated with the pubic symphysis, the remaining caudal fistula was removed from the body surface with a small spindle-shaped incision around the fistula opening. Finally, the sinus was completely resected, with confirmation from both the cranial side and dorsal side of the pubic symphysis. We were able to perform complete resection of the CPS with good visibility and without any peritoneal damage. There were no intraoperative complications. His postoperative course was uneventful during the 1-year follow-up. CONCLUSIONS: The TEP approach may be feasible for the resection of a CPS and may allow safe and secure resection due to good visibility, even in pediatric patients.

Stability of probiotics with antibiotics via gastric tube by simple suspension method: An in vitro study.

Data on the stability of probiotics with antibiotics delivered via gastric tube using the simple suspension method (SSM) are limited. Therefore, we investigated bacterial survivability in probiotics treated with antibiotics prepared by the SSM in vitro. Probiotics and antibiotics were suspended in 20 mL of sterilized hot water (55 °C) and then 1-mL of the suspensions were taken each at 10, 60, 120, 180 and 360 min. Thereafter, the samples were inoculated on 3 media and cultured at 37 °C for 24 h. Survival of probiotic strains was measured in colony-forming units. The growth of Clostridium butyricum did not change without antibiotics at all experimental times, but in the case of Enterococcus faecium tended to increase. On the other hand, the viable bacterial number of C. butyricum was decreased significantly by treatment with cefdinir, tosufloxacin, clarithromycin, or azithromycin, but was not altered by levofloxacin, minocycline, or vancomycin. The viable bacterial number of E. faecium was significantly decreased by treatment with tosufloxacin, levofloxacin, minocycline, vancomycin, or azithromycin, and was significantly increased by clarithromycin. In conclusion, our results suggest that the efficacy of probiotic therapies might be reduced by the SSM when specific antibiotics are used. Moreover, antibiotics might inhibit probiotic growth, although some probiotics are spore-forming and have high minimum inhibitory concentrations. Additionally, early administration of non-spore-forming bacteria might be desirable. Therefore, when patients are administered therapy combining probiotics and antibiotics by the SSM, we should consider the characteristics of the probiotics and the administration times.

Diversities and similarities in pH dependency among bacterial NhaB-like Na+/H+ antiporters.

NhaB-like antiporters were the second described class of Na(+)/H(+) antiporters, identified in bacteria more than 20 years ago. While nhaB-like gene sequences have been found in a number of bacterial genomes, only a few of the NhaB-like antiporters have been functionally characterized to date. Although earlier studies have identified a few pH-sensitive and -insensitive NhaB-like antiporters, the mechanisms that determine their pH responses still remain elusive. In this study, we sought to investigate the diversities and similarities among bacterial NhaB-like antiporters, with particular emphasis on their pH responsiveness. Our phylogenetic analysis of NhaB-like antiporters, combined with pH profile analyses of activities for representative members of several phylogenetic groups, demonstrated that NhaB-like antiporters could be classified into three distinct types according to the degree of their pH dependencies. Interestingly, pH-insensitive NhaB-like antiporters were only found in a limited proportion of enterobacterial species, which constitute a subcluster that appears to have diverged relatively recently among enterobacterial NhaB-like antiporters. Furthermore, kinetic property analyses of NhaB-like antiporters at different pH values revealed that the degree of pH sensitivity of antiport activities was strongly correlated with the magnitude of pH-dependent change in apparent Km values, suggesting that the dramatic pH sensitivities observed for several NhaB-like antiporters might be mainly due to the significant increases of apparent Km at lower pH. These results strongly suggested the possibility that the loss of pH sensitivity of NhaB-like antiporters had occurred relatively recently, probably via accumulation of the mutations that impair pH-dependent change of Km in the course of molecular evolution.

Critical involvement of the E373-D434 region in the acid sensitivity of a NhaB-type Na(+)/H(+) antiporter from Vibrio alginolyticus.

It has been well established that VaNhaB, a NhaB-type Na(+)/H(+) antiporter found in Vibrio alginolyticus, exhibits a striking acid sensitivity. However, the molecular basis of the pH-dependent regulatory mechanism of the antiport activity is yet to be investigated. In this study, we generated various chimeric proteins composed of VaNhaB and a pH insensitive ortholog found in Escherichia coli (EcNhaB) and analyzed the pH responses of their Na(+)/H(+) antiport activities to search for the key residues or domains that are involved in the pH sensitivity of VaNhaB. Our results revealed the significant importance of a stretch of amino acid residues within the loop 8-loop 9 regions (E373-D434) responsible for the acid sensitivity of VaNhaB, along with the possible involvement of other unidentified residues that are widely spread in the primary structure of VaNhaB. Moreover, we demonstrated that the E373-D434 region of VaNhaB was able to confer some degree of acid sensitivity on our pH insensitive chimeric antiporter that is mainly composed of EcNhaB except for seven amino acid substitutions at the N-terminal end. This result strongly suggested the possibility that the E373-D434 region is able to act, at least partially, as machinery that diminishes the activity of the NhaB-type antiporter at an acidic pH.

Genome comparison and phylogenetic analysis of Orientia tsutsugamushi strains.

Orientia tsutsugamushi (OT) is an obligate intracellular bacterium belonging to the family Rickettsiaceae and is the causative agent of scrub typhus, or Tsutsugamushi disease. The complete genome sequences of two OT strains (Boryong and Ikeda) have recently been determined. In the present study, we performed a fine genome sequence comparison of these strains. Our results indicate that although the core gene set of the family Rickettsiaceae is highly conserved between the two strains, a common set of repetitive sequences have been explosively amplified in both genomes. These amplified repetitive sequences have induced extensive genome shuffling and duplications and deletions of many genes. On the basis of the results of the genome sequence comparison, we selected 11 housekeeping genes and carried out multilocus sequence analysis of OT strains using the nucleotide sequences of these genes. This analysis revealed for the first time the phylogenetic relationships of representative OT strains. Furthermore, the results suggest the presence of an OT lineage with higher potential for virulence, which may explain the clinical and epidemiological differences between 'classic' and 'new' types of Tsutsugamushi disease in Japan.

pH-dependent regulation of the multi-subunit cation/proton antiporter Pha1 system from Sinorhizobium meliloti.

The pha1 gene cluster (pha1A'-G) of Sinorhizobium meliloti has previously been characterized as a necessary component for proper invasion into plant root tissue. It has been suggested to encode a multi-subunit K(+)/H(+) antiporter, since mutations in the pha1 region rendered S. meliloti cells sensitive to K(+) and alkali, and because there is high amino acid sequence similarity to previously characterized multi-subunit cation/H(+) antiporters (Mrp antiporters). However, the detailed transport properties of the Pha1 system are yet to be determined. Interestingly, most of the Mrp antiporters are highly selective for Na(+), unlike the Pha1 system. Here, we report the functional expression of the Pha1 system in Escherichia coli and the measurement of cation/H(+) antiport activity. We showed that the Pha1 system is indeed a K(+)/H(+) antiporter with a pH optimum under mildly alkaline conditions. Moreover, we found that the Pha1 system can transport Na(+); this was unexpected based on previous phenotypic analyses of pha1 mutants. Furthermore, we demonstrated that the cation selectivity of the Pha1 system was altered when the pH was lowered from the optimum. The downregulation of Na(+)/H(+) and K(+)/H(+) antiport activities upon acidic shift appeared to occur via different processes, which might indicate the presence of distinct mechanisms for the regulation of the K(+)/H(+) and Na(+)/H(+) antiport activities of the Pha1 system.

The Whole-genome sequencing of the obligate intracellular bacterium Orientia tsutsugamushi revealed massive gene amplification during reductive genome evolution.

Scrub typhus ('Tsutsugamushi' disease in Japanese) is a mite-borne infectious disease. The causative agent is Orientia tsutsugamushi, an obligate intracellular bacterium belonging to the family Rickettsiaceae of the subdivision alpha-Proteobacteria. In this study, we determined the complete genome sequence of O. tsutsugamushi strain Ikeda, which comprises a single chromosome of 2 008 987 bp and contains 1967 protein coding sequences (CDSs). The chromosome is much larger than those of other members of Rickettsiaceae, and 46.7% of the sequence was occupied by repetitive sequences derived from an integrative and conjugative element, 10 types of transposable elements, and seven types of short repeats of unknown origins. The massive amplification and degradation of these elements have generated a huge number of repeated genes (1196 CDSs, categorized into 85 families), many of which are pseudogenes (766 CDSs), and also induced intensive genome shuffling. By comparing the gene content with those of other family members of Rickettsiacea, we identified the core gene set of the family Rickettsiaceae and found that, while much more extensive gene loss has taken place among the housekeeping genes of Orientia than those of Rickettsia, O. tsutsugamushi has acquired a large number of foreign genes. The O. tsutsugamushi genome sequence is thus a prominent example of the high plasticity of bacterial genomes, and provides the genetic basis for a better understanding of the biology of O. tsutsugamushi and the pathogenesis of 'Tsutsugamushi' disease.

Potassium/proton antiport system of Escherichia coli.

The intracellular level of potassium (K(+)) in Escherichia coli is regulated through multiple K(+) transport systems. Recent data indicate that not all K(+) extrusion system(s) have been identified (15). Here we report that the E. coli Na(+) (Ca(2+))/H(+) antiporter ChaA functions as a K(+) extrusion system. Cells expressing ChaA mediated K(+) efflux against a K(+) concentration gradient. E. coli strains lacking the chaA gene were unable to extrude K(+) under conditions in which wild-type cells extruded K(+). The K(+)/H(+) antiporter activity of ChaA was detected by using inverted membrane vesicles produced using a French press. Physiological growth studies indicated that E. coli uses ChaA to discard excessive K(+), which is toxic for these cells. These results suggest that ChaA K(+)/H(+) antiporter activity enables E. coli to adapt to K(+) salinity stress and to maintain K(+) homeostasis.

Expression of the regulatory protein LndI for landomycin E production in Streptomyces globisporus 1912 is controlled by the availability of tRNA for the rare UUA codon.

The gene lndI encodes the activator of landomycin biosynthesis. The utilization of LndI-EGFP fusions led us to investigate the temporal pattern of this gene expression and demonstrated the delay between lndI transcription and translation. The TTA codon in lndI is thought to be the reason for this delay. The replacement of TTA with CTC cancelled the pause between lndI transcription and the translation. The wild-type of the lndI gene is not expressed in the Streptomyces coelicolor bldA- mutant strain, indicating the importance of the bldA tRNA in its mRNA translation.

Cloning, functional expression and primary characterization of Vibrio parahaemolyticus K+/H+ antiporter genes in Escherichia coli.

The regulation of internal Na(+) and K(+) concentrations is important for bacterial cells, which, in the absence of Na(+) extrusion systems, cannot grow in the presence of high external Na(+). Likewise, bacteria require K(+) uptake systems when the external K(+) concentration becomes too low to support growth. At present, we have little knowledge of K(+) toxicity and bacterial outward-directed K(+) transport systems. We report here that high external concentrations of K(+) at alkaline pH are toxic and that bacteria require K(+) efflux and/or extrusion systems to avoid excessive K(+) accumulation. We have identified the first example of a bacterial K(+)(specific)/H(+) antiporter, Vp-NhaP2, from Vibrio parahaemolyticus. This protein, a member of the cation : proton antiporter-1 (CPA1) family, was able to mediate K(+) extrusion from the cell to provide tolerance to high concentrations of external KCl at alkaline pH. We also report the discovery of two V. parahaemolyticus Na(+)/H(+) antiporters, Vp-NhaA and Vp-NhaB, which also exhibit a novel ion specificity toward K(+), implying that they work as Na(+)(K(+))/H(+) exchangers. Furthermore, under specific conditions, Escherichia coli was able to mediate K(+) extrusion against a K(+) chemical gradient, indicating that E. coli also possesses an unidentified K(+) extrusion system(s).

Survival of two Orientia tsutsugamushi bacterial strains that infect mouse macrophages with varying degrees of virulence.

Orientia tsutsugamushi, an intracellular parasitic bacterium, comprises numerous strains of differing virulence. When BALB/c mice were infected intraperitoneally with this pathogen, a virulent strain known as Karp was found to multiply in the intraperitoneal macrophages and kill the mouse. In contrast, an avirulent strain, Kuroki, was shown to invade macrophages but be eliminated from the cells, allowing mouse survival. O. tsutsugamushi invades its host cell cytoplasm through phagocytosis and disruption of phagosomal membranes but some bacteria are then killed by phago-lysosomes within 1h of infection. Microscopic observations could not differentiate the Karp and Kuroki strains during entry and subsequent cell killing by phago-lysosomes. However, the Kuroki cells failed to divide and were markedly deformed following cytoplasmic invasion at several days post-infection. These findings suggest that macrophages have a mechanism to eliminate O. tsutsugamushi in the cytoplasm, if the invading bacteria escape phagosomal clearance, and that it is this mechanism that Kuroki does not survive. Additionally, significant levels of nitric oxide (NO) are produced in macrophages by Kuroki, but not by Karp. An NO synthase inhibitor, however, does not increase the growth of Kuroki, suggesting that NO is induced in a strain-dependent manner but does not effect proliferation.

Generation of Streptomyces globisporus SMY622 strain with increased landomycin E production and it's initial characterization.

Landomycin E (LaE) overproducing strain Streptomyces globisporus SMY6222 has been developed using UV induced mutagenesis and selection for streptomycin resistance. SMY622 has been shown by HPLC to produce 200-fold higher amounts of LaE when comparing with parental strain. The levels of transcription of regulatory gene lndI and oxygenase gene lndE are two times higher in the mutant than in the wild type. Gene rpsL for ribosomal protein S12 from SMY622 was shown to contain point mutation K43R. Possible reasons for increased LaE synthesis in SMY622 are discussed.