Validation of the cell culture monitoring using a Raman spectroscopy calibration model developed with artificially mixed samples and investigation of model learning methods using initial batch data.

The development of calibration models using Raman spectra data has long been challenged owing to the substantial time and cost required for robust data acquisition. To reduce the number of experiments involving actual incubation, a calibration model development method was investigated by measuring artificially mixed samples. In this method, calibration datasets were prepared using spectra from artificially mixed samples with adjusted concentrations based on design of experiments. The precision of these calibration models was validated using the actual cell culture sample. The results showed that when the culture conditions were unchanged, the root mean square error of prediction (RMSEP) of glucose, lactate, and antibody concentrations was 0.34, 0.33, and 0.25 g/L, respectively. Even when variables such as cell line or culture media were changed, the RMSEPs of glucose, lactate, and antibody concentrations remained within acceptable limits, demonstrating the robustness of the calibration models with artificially mixed samples. To further improve accuracy, a model training method for small datasets was also investigated. The spectral pretreatment conditions were optimized using error heat maps based on the first batch of each cell culture condition and applied these settings to the second and third batches. The RMSEPs improved for glucose, lactate, and antibody concentration, with values of 0.44, 0.19, and 0.18 g/L under constant culture conditions, 0.37, 0.12, and 0.12 g/L for different cell lines, and 0.26, 0.40, and 0.12 g/L when the culture media was changed. These results indicated the efficacy of calibration modeling with artificially mixed samples for actual incubations under various conditions.

Development of Raman Calibration Model Without Culture Data for In-Line Analysis of Metabolites in Cell Culture Media.

In this study, we developed a method to build Raman calibration models without culture data for cell culture monitoring. First, Raman spectra were collected and then analyzed for the signals of all the mentioned analytes: glucose, lactate, glutamine, glutamate, ammonia, antibody, viable cells, media, and feed agent. Using these spectral data, the specific peak positions and intensities for each factor were detected. Next, according to the design of the experiment method, samples were prepared by mixing the above-mentioned factors. Raman spectra of these samples were collected and were used to build calibration models. Several combinations of spectral pretreatments and wavenumber regions were compared to optimize the calibration model for cell culture monitoring without culture data. The accuracy of the developed calibration model was evaluated by performing actual cell culture and fitting the in-line measured spectra to the developed calibration model. As a result, the calibration model achieved sufficiently good accuracy for the three components, glucose, lactate, and antibody (root mean square errors of prediction, or RMSEP = 0.23, 0.29, and 0.20 g/L, respectively). This study has presented innovative results in developing a culture monitoring method without using culture data, while using a basic conventional method of investigating the Raman spectra of each component in the culture media and then utilizing a design of experiment approach.

Tension-free thoracoscopic repair of congenital diaphragmatic hernia combined with a percutaneous extracorporeal closure technique: how to do it.

PURPOSE: Thoracoscopic repair (TR) of congenital diaphragmatic hernia (CDH) is associated with a higher recurrence rate than the conventional open method. We evaluated the effectiveness of our strategy for quality improvement, named "tension-free TR of CDH". METHODS: The subjects of this retrospective analysis were 11 consecutive patients with CDH who underwent TR at our hospital between 2017 and 2021. Tension-free TR of CDH included the proactive use of an oversized patch for dome-shaped reconstruction and gapless suturing. We developed a percutaneous extracorporeal closure technique for secure suturing using a commercially available needle. RESULTS: Patch repair was performed in 8 (73%) patients and none required conversion to open surgery because of technical difficulties. Recurrence developed in one patient (9%), who underwent successful reoperation via TR. All patients had an uneventful postoperative course. CONCLUSION: Tension-free TR combined with extracorporeal closure could reduce the difficulty of suturing and the risk of recurrence of CDH.

Successful salvage of torsion testis by means of intraoperative indocyanine green fluorescence imaging.

BACKGROUND: Testicular torsion (TT) is common surgical emergency that requires early diagnosis and immediate intervention within 6 h since its onset to salvage the testis. However, the decision was made only by the surgeon's experience whether it has to be resected or not. Recently, indocyanine green (ICG) has become an excellent tool to identify biliary and vascular anatomy, and assess perfusion abnormalities in tissues. In this case report, we successfully salvaged the twisted testis, since the testicular blood perfusion was confirmed by means of intraoperative ICG (IICG) fluorescence imaging. CASE PRESENTATION: A 14-year-old healthy male patient presented due to acute left testicular pain. The patient was diagnosed with TT and had immediate surgery. Macroscopically, the testis had stagnant blood flow and appeared to be dark colored. After manual detorsion, the testis remained cyanotic and with macroscopically poor blood flow. ICG angiography was performed under near-infrared light by laparoscopic camera to assess the perfusion of the affected testicle. An excellent ICG signal appeared after 45 s in the testis, and decision was made to be preserved. Therefore, left orchidopexy was performed to complete the operation. The patient had a good postoperative course and was discharged the day after surgery. Six months later, the testis did not show any shrinkage, and both sides of the testis showed the same size without any consequences. CONCLUSION: The blood flow in the testis was visually confirmed during the IICG fluorescence method. ICG fluorescence imaging may become an effective alternative to evaluate whether a testis can be preserved following TT.

Factors Associated With Adverse Outcomes Following Duodenal Atresia Surgery in Neonates: A Retrospective Study.

Objectives There is limited evidence on the infants' postoperative complications who have undergone surgical repair of duodenal atresia and stenosis. This study aimed to identify the factors associated with poor surgical outcomes after the initial repair. Methods We retrospectively reviewed the data of 82 patients who underwent surgery for duodenal atresia and stenosis between January 1994 and December 2013 at our institution. Gestational age, birth weight, fetal growth, and other associated anomalies were recorded. Multivariate regression analyses were used to identify the factors associated with surgical outcomes, including postoperative complications and time to full oral intake. Results The median gestational age was 37.6 weeks, with 30 (37%) preterm (<37 weeks) and 11 (13%) early preterm (<34 weeks) infants. The median birth weight was 2531 g, with 27 (33%) patients < 2000 g and 10 (12%) patients < 1500 g. Postoperative surgical complications were identified in 18 (22%) cases, of which 12 (15%) required additional operations. Multivariate regression analysis revealed that a combination of very low birth weight (<1500 g) and early preterm was significantly associated with both surgical and non-surgical postoperative complications (p = 0.0028 and 0.021, respectively) and a prolonged time to full oral intake postoperatively (p = 0.013). Conclusion Very low birth weight and early preterm were significantly associated with postoperative complications and a prolonged time to full oral intake.

Can plain chest X-ray replace computed tomography for the follow-up of children who have undergone the Nuss procedure?

PURPOSE: To establish whether new indices on plain chest X-ray (CXR) can replace those on computed tomography (CT) for the follow-up of children who have undergone the Nuss procedure. METHODS: The subjects of this retrospective study were 45 children who underwent the Nuss procedure between 2000 and 2016. The Haller index (HI) was measured by preoperative CT. Preoperative and postoperative chest deformities were evaluated by two CXR measurements: the concave rate on the lateral view (CR; the depth of the concavity divided by the anterior-posterior diameter of the rib cage) and the tracheal bifurcation angle (TBA) on the anterior view. Data are expressed as the median with range. RESULTS: The median age and HI of the children, when they underwent the Nuss procedure, was 9.3 (3.8-17.3) years and 4.5 (3.2-10.1), respectively. The preoperative CR was correlated significantly with the HI. The postoperative CR was significantly lower than the preoperative CR [pre: 0.17 (0.08-0.37), post: 0.09 (0.01-0.18), p < 0.05], and the low value was sustained after bar removal. The TBA decreased significantly after the Nuss procedure from 74.2° (55-104) preoperatively to 65.0° (45-92) postoperatively (p < 0.05). CONCLUSIONS: These results suggest that CXR can replace CT for the follow-up of patients after the Nuss procedure, with lower radiation exposure.

Parotid Gland Non-Sebaceous Lymphadenoma: A Case Report.

We here report a rare salivary gland tumor, a non-sebaceous lymphadenoma (NSLA), in the parotid gland with positive technetium-99m (99mTc) pertechnetate scintigraphy and cervical masses that was difficult to distinguish from malignant lymphoma preoperatively. A 65-year-old Japanese woman presented to our institution with a painless left parotid mass that had been gradually enlarging for three months. Computed tomography and magnetic resonance imaging showed a solid mass (30 mm) in the tail of the left parotid gland with three swellings (each 10 mm) on the periphery of the parotid gland. A low grade malignant lymphoma was diagnosed by fine needle aspiration cytology. Gallium-67 scintigraphy showed mild positivity. On 99mTc pertechnetate scintigraphy, the mild accumulation remained after oral stimulation with a mixture of ascorbic acid and calcium pantothenate. The patient underwent superficial parotidectomy with removal of the cervical masses. Our findings in this patient suggest that 99mTc positivity may constitute a clue to the preoperative diagnosis of NSLA that has pathologically overlapping sites with Warthin tumor.

Learning curve for the thoracoscopic repair of esophageal atresia with tracheoesophageal fistula.

AIM: Thoracoscopic repair (TR) of esophageal atresia with tracheoesophageal fistula (EA/TEF) remains a considerable challenge, even for the most experienced pediatric surgeons. The aim of this study is to report the outcomes of our experience with TR of EA/TEF and to determine the learning curve for this procedure. METHODS: Eleven consecutive cases that had undergone TR of EA/TEF at our institutes were included in this study. The medical charts were reviewed retrospectively. To determine the learning curve for TR of EA/TEF, a logarithmic curve-fitting analysis was performed. The data were expressed as medians with ranges. RESULTS: The median age and birth weight were 1 day (range, 1-3 days) and 2.8 kg (range, 2.5-3.7 kg), respectively. TR was completed in all cases without any complications. The median operative time was 230 min (range, 164-383 min). There were no cases of anastomotic leakage. One patient with a long gap required repeated balloon dilatation for refractory anastomotic stricture. No mortality or recurrence of tracheoesophageal fistula occurred. The operative time was significantly longer in patients with a long gap (>20 mm) than in those with a shorter gap. Once the three cases with a long gap had been excluded, the operative time decreased as the number of treated cases increased. The relationship between the operative time and case number fit a logarithmic function curve well (operative time in minutes = 300 - 62 × log (case number), R2  = 0.8359, P = 0.0015). CONCLUSIONS: Our results suggest that TR of EA/TEF is a safe procedure. It has a considerable learning curve, but requires advanced endoscopic surgical skills.

Onset ages of hepatopulmonary syndrome and pulmonary hypertension in patients with biliary atresia.

PURPOSE: Hepatopulmonary syndrome (HPS) and portopulmonary hypertension (PoPH) are extrahepatic complications of biliary atresia (BA). Their detection is sometimes delayed, which may result in missed opportunities for liver transplantation. The aim of this study was to determine the onset ages of HPS and PoPH in BA patients. METHODS: BA patients followed at our institution were identified. Patients visited our clinic for routine blood work, as well as regular electrocardiography, chest X-rays, and arterial blood gas tests. Lung perfusion scintigraphy and cardiac ultrasound were performed to diagnose HPS. Cardiac catheterization was conducted to diagnose PoPH. RESULTS: The study population consisted of 88 BA patients. The median follow-up duration was 11.6 years (range 0.8-26.0 years). Six patients (6.8%) developed HPS and three patients (3.4%) developed PoPH. The median age of onset of HPS was significantly younger than that of PoPH (HPS: 4 years, PoPH: 15 years, P < 0.019). Two patients (66%) with PoPH died, while all patients with HPS survivied. CONCLUSION: The onset of HPS was significantly earlier than that of PoPH. The mortality rate was high in patients with PoPH. Teenagers with BA should receive routine cardiac echocardiograms to detect PH in its early stages.

Magnetic compression anastomosis for postoperative biliary atresia.

We report a case of successful magnetic compression anastomosis (MCA) for obstructed cyst-jejunostomy in a young woman who had undergone surgery for type 1 biliary atresia (BA) on day 78 of life. A 16-year-old girl was admitted with obstructive jaundice. Jaundice resolved with percutaneous trans-hepatic cholangiodrainage (PTCD) but contrast medium injected from the PTCD tube did not flow through the anastomosis. Magnets were placed on each side of the anastomosis, in the cyst and the jejunum, to compress the partition. On postoperative day (POD) 6, the anastomosis was recanalized and the PTCD tube placed trans-anastomotically until POD 245. The patient remained free from jaundice after removal of the PTCD tube. MCA can be a useful and less invasive procedure for treating biliary tract anastomotic obstruction in patients with BA.

Long-term morbidity in adolescents and young adults with surgically treated esophageal atresia.

PURPOSE: To investigate the long-term morbidity of surgically treated esophageal atresia (EA) in adolescents and young adults and establish whether these long-term morbidities are affected by the type of EA. PATIENTS AND METHODS: We reviewed the medical records, including backgrounds and associated conditions, of 69 long-term survivors of EA, aged >15 years. The long-term morbidities included neurodevelopmental abnormality, nutritional impairment (short height <-2SD, low BMI <18.5), subjective symptoms, and musculoskeletal deformities. Comparisons of the results were made between Gross A-type EA (n = 6) and Gross C-type EA (n = 63). RESULTS: All patients underwent esophageal anastomosis without esophageal replacement. Cardiac anomalies and long gap were present in 26 and 18%, respectively. Esophageal dilatation, fundoplication, and aortopexy were performed in 40, 34, and 18%, respectively. The incidence of long gap and esophageal stenosis was higher in Gross A-type EA than in Gross C-type EA. The long-term morbidities included neurodevelopmental abnormality (13%), nutritional impairment (62%: as short height in 34% and as low BMI in 46%), subjective symptoms (14%), and musculoskeletal deformities (59%). There were no differences in the long-term morbidities between Gross A and Gross C. CONCLUSIONS: The incidence of the long-term morbidities is high in adolescents and young adults, regardless of the type of EA. Early detection of morbidity is important to improve the long-term outcomes of EA.

Two Different Functions of Connexin43 Confer Two Different Bone Phenotypes in Zebrafish.

Fish remain nearly the same shape as they grow, but there are two different modes of bone growth. Bones in the tail fin (fin ray segments) are added distally at the tips of the fins and do not elongate once produced. On the other hand, vertebrae enlarge in proportion to body growth. To elucidate how bone growth is controlled, we investigated a zebrafish mutant, steopsel (stp(tl28d)). Vertebrae of stp(tl28d) (/+) fish look normal in larvae (∼30 days) but are distinctly shorter (59-81%) than vertebrae of wild type fish in adults. In contrast, the lengths of fin rays are only slightly shorter (∼95%) than those of the wild type in both larvae and adults. Positional cloning revealed that stp encodes Connexin43 (Cx43), a connexin that functions as a gap junction and hemichannel. Interestingly, cx43 was also identified as the gene causing the short-of-fin (sof) phenotype, in which the fin ray segments are shorter but the vertebrae are normal. To identify the cause of this difference between the alleles, we expressed Cx43 exogenously in Xenopus oocytes and performed electrophysiological analysis of the mutant proteins. Gap junction coupling induced by Cx43(stp) or Cx43(sof) was reduced compared with Cx43-WT. On the other hand, only Cx43(stp) induced abnormally high (50× wild type) transmembrane currents through hemichannels. Our results suggest that Cx43 plays critical and diverse roles in zebrafish bone growth.

Long-term outcomes of four patients with tracheal agenesis who underwent airway and esophageal reconstruction.

PURPOSE: The aim of this study was to evaluate the long-term outcomes of four patients with tracheal agenesis who underwent airway and esophageal/alimentary reconstruction. MATERIALS AND METHODS: We reviewed the medical records of four long-term survivors of tracheal agenesis and collected the following data: age, sex, type of tracheal agenesis, method of reconstruction, nutritional management, and physical and neurological development. RESULTS: The patients consisted of three boys and one girl, who ranged in age from 77 to 109months. The severity of their condition was classified as Floyd's type I (n=2), II (n=1), or III (n=1). Mechanical respiratory support was not necessary in any of the cases. Esophageal/alimentary reconstruction was performed using the small intestine (n=2), a gastric tube (n=1), and the esophagus (n=1). The age at esophageal reconstruction ranged from 41 to 55months. All of the cases required enteral nutrition via gastrostomy. Three of the patients were able to swallow a small amount of liquid and one was able to take pureed food orally. The physical development of the subjects was moderately delayed-borderline in childhood. Neurological development was normal in two cases and slightly delayed in two cases. CONCLUSIONS: None of the long-term survivors of tracheal agenesis required the use of an artificial respirator, and their development was close to normal. Future studies should aim to elucidate the optimal method for performing esophageal reconstruction to allow tracheal agenesis patients to achieve their full oral intake.

Treatment and ovarian preservation in children with ovarian tumors.

BACKGROUND/PURPOSE: Ovarian preservation is desirable in children with ovarian tumors. However, the diagnostic and treatment strategies are heterogeneous. The aim of this study was to investigate the management and preservation of ovarian tissue in order to identify the factors associated with ovarian preservation. METHODS: Thirty-seven patients (41 ovaries) were surgically treated for ovarian tumors. Four cases were bilateral. The data on the patient symptoms at presentation, imaging, treatment, outcome, pathology, and status of ovarian preservation were retrospectively analyzed for each patient. RESULTS: Histological examinations revealed 25 mature teratomas, 5 immature teratomas, 5 cystadenomas, 2 dysgerminomas, and other tumors. Ovarian torsion occurred in 16 ovaries (39%). Ovary-sparing surgery was performed in 22 ovaries (53.7%). Successful ovarian preservation was significantly associated with a smaller tumor size, benign pathology, and a lower degree of torsion (P<0.01). CONCLUSIONS: Because the prognosis was favorable in most cases, the preservation of fertility and gonadal function should be a goal in the surgical treatment of ovarian tumors. We recommend ovary-sparing surgery as the first-line treatment for all pediatric ovarian tumors other than those that are preoperatively diagnosed as being malignant and those in which emergent surgical intervention is indicated owing to the suspicion of ovarian torsion.

Rotating pigment cells exhibit an intrinsic chirality.

In multicellular organisms, cell properties, such as shape, size and function are important in morphogenesis and physiological functions. Recently, 'cellular chirality' has attracted attention as a cellular property because it can cause asymmetry in the bodies of animals. In recent in vitro studies, the left-right bias of cellular migration and of autonomous arrangement of cells under some specific culture conditions were discovered. However, it is difficult to identify the molecular mechanism underlying their intrinsic chirality because the left-right bias observed to date is subtle or is manifested in the stable orientation of cells. Here, we report that zebrafish (Danio rerio) melanophores exhibit clear cellular chirality by unidirectional counterclockwise rotational movement under isolated conditions without any special settings. The chirality is intrinsic to melanophores because the direction of the cellular rotation was not affected by the type of extracellular matrix. We further found that the cellular rotation was generated as a counter action of the clockwise movement of actin cytoskeleton. It suggested that the mechanism that directs actin cytoskeleton in the clockwise direction is pivotal for determining cellular chirality.

In vitro analysis suggests that difference in cell movement during direct interaction can generate various pigment patterns in vivo.

Pigment patterns of organisms have invoked strong interest from not only biologists but also, scientists in many other fields. Zebrafish is a useful model animal for studying the mechanism of pigment pattern formation. The zebrafish stripe pattern is primarily two types of pigment cells: melanophores and xanthophores. Previous studies have reported that interactions among these pigment cells are important for pattern formation. In the recent report, we found that the direct contact by xanthophores induces the membrane depolarization of melanophores. From analysis of jaguar mutants, it is suggested that the depolarization affects the movements of melanophores. To analyze the cell movement in detail, we established a unique in vitro system. It allowed us to find that WT xanthophores induced repulsive movement of melanophores through direct contact. The xanthophores also chased the melanophores. As a result, they showed run-and-chase movements. We also analyzed the cell movement of pigment cells from jaguar and leopard mutants, which have fuzzy stripes and spot patterns, respectively. jaguar cells showed inhibited run-and-chase movements, and leopard melanophores scarcely showed repulsive response. Furthermore, we paired mutant and WT cells and showed which of the melanophores and xanthophores have responsibility for the altered cell movements. These results suggested that there is a correspondence relationship between the cell movements and pigment patterns. The correspondence relationship highlighted the importance of the cell movements in the pattern formation and showed that our system is a quite useful system for future study in this field.

Hedgehog signal inhibitors suppress the invasion of human rhabdomyosarcoma cells.

PURPOSE: In the treatment of rhabdomyosarcoma (RMS), invasion and metastasis remain the most critical determinants of resectability and survival. The objective of this study was to determine whether Hedgehog (Hh) signaling plays a role in the invasion of RMS. METHODS: Two kinds of specific Hh signaling inhibitors, cyclopamine and forskolin, were used to suppress activated Hh signals in three RMS cell lines. The effects of the Hh signaling inhibitors on tumor cell invasion and motility were investigated using Matrigel invasion assays and wound closure assays, respectively. RESULTS: The number of invaded cells counted in six random microscopic fields in the Matrigel chambers was significantly decreased by both cyclopamine and forskolin in every RMS cell line. Furthermore, the wound closure assays revealed that a blockade of the Hh signaling pathway by the Hh inhibitors strongly impairs RMS cell motility, as visualized by the delayed closure of the gaps generated in the cultured cell monolayers of the three RMS cell lines. CONCLUSIONS: Both the invasive capacity and motility of RMS cells are significantly suppressed by Hh signaling inhibitors, demonstrating that the Hh pathway plays an important role in the invasion of RMS. Hh inhibitors may provide a new paradigm for the treatment of RMS.

Caudal appendage derived from a vestigial remnant of the tailgut.

The presence of smooth muscle at the basal portion of a caudal appendage is very rare. We report a 3-month-old girl in which a caudal appendage is associated with smooth muscle bundles at the perianal region. Immunohistochemistry was performed for NCAM (neural cell adhesion molecule) to identify smooth muscle. NCAM immunoreactivity was observed within the presumptive circular and/or longitudinal muscle layers of the muscularis propria. NCAM is expressed by smooth muscle during the early stages of human embryonic gut development, suggesting that the caudal appendage in the present case may be derived from a tailgut remnant.

Single-port laparoscopic repair of an epigastric hernia using an epidural needle.

INTRODUCTION: Epigastric hernia is a rare form of ventral hernia, occurring along the linea alba anywhere from the xiphoid process to the umbilicus. PRESENTATION OF CASE: We present the case of a 19-month-old boy with an epigastric hernia who underwent a single-port laparoscopic repair using an epidural needle. A nonabsorbable suture was threaded through an epidural needle, with one end of the suture threaded back through the needle tip to make a loop. The loop-shaped suture was left in the abdominal cavity through the rectus muscle at the site of the defect. Another suture was inserted into the looped suture through the opposite rectus muscle. The loop was pulled taut and the defect was closed by tying the suture extracorporeally with a subcutaneous knot. DISCUSSION: In epigastric hernia, the surgical technique involves overlapping muscle layers, and currently laparoscopic surgery is introduced to repair the hernia defect. Laparoscopy is a minimally invasive method of repairing epigastric hernias. CONCLUSION: The epidural needle proved a simple and cosmetically acceptable device with which to close the epigastric hernia defect.

Closure of a tracheocutaneous fistula by two hinged turnover skin flaps and a muscle flap: A case report.

INTRODUCTION: Tracheocutaneous fistula is a complication of tracheostomy. Tracheocutaneous fistulectomy followed by primary closure carries a high possibility of complications. PRESENTATION OF CASE: An 11-year-old boy underwent surgery to repair a tracheocutaneous fistula, using skin and muscle flaps. A vertical incision was made around the fistula and 3 skin flaps were prepared: 2 hinge flaps, and 1 to cover the skin defect (advanced flap). The 2 hinged turnover flaps were invaginated by multiple layered sutures, and a strap muscle flap was placed over the resulting tracheal closure. An advanced skin flap was used to cover the area of the previous defect. The patient was extubated immediately after surgery. He was discharged on the sixth postoperative day without tracheal leakage or subcutaneous emphysema. The patient is currently doing well, with no respiratory symptoms and no recurrence at the postoperative 5 months. DISCUSSION: Our technique is minimally invasive and has a low risk of lumen stenosis, other complications, or recurrence. CONCLUSION: This technique demonstrates the multiple-layered closure of a tracheocutaneous fistula, using skin flaps and a muscle flap.