Water-in-oil droplet-mediated method for detecting and isolating infectious bacteriophage particles via fluorescent staining.

Bacteriophages are the most abundant entities on Earth. In contrast with the number of phages considered to be in existence, current phage isolation and screening methods lack throughput. Droplet microfluidic technology has been established as a platform for high-throughput screening of biological and biochemical components. In this study, we developed a proof-of-concept method for isolating phages using water-in-oil droplets (droplets) as individual chambers for phage propagation and co-cultivating T2 phage and their host cell Escherichia coli within droplets. Liquid cultivation of microbes will facilitate the use of microbes that cannot grow on or degrade agar as host cells, ultimately resulting in the acquisition of phages that infect less known bacterial cells. The compartmentalizing characteristic of droplets and the use of a fluorescent dye to stain phages simultaneously enabled the enumeration and isolation of viable phage particles. We successfully recultivated the phages after simultaneously segregating single phage particles into droplets and inoculating them with their host cells within droplets. By recovering individual droplets into 96-well plates, we were able to isolate phage clones derived from single phage particles. The success rate for phage recovery was 35.7%. This study lays the building foundations for techniques yet to be developed that will involve the isolation and rupturing of droplets and provides a robust method for phage enumeration and isolation.

Genome editing is induced in a binary manner in single human cells.

Even when precise nucleotide manipulations are intended, the outcomes of genome editing can be diverse, often including random insertions and deletions. The combinations and frequencies of these different outcomes in single cells are critical not only in the generation of genetically modified cell lines but also in the evaluation of the clinical effects of genome editing therapies. However, current methods only analyze cell populations, not single cells. Here, we utilized the Single Particle isolation System (SPiS) for the efficient isolation of single cells to systematically analyze genome editing results in individual human cultured cells. As a result, we discovered that genome editing induction has a binary nature, that is, the target alleles of cells tend to be all edited or not edited at all. This study enhances our understanding of the induction pattern of genome editing and provides a new strategy to analyze genome editing outcomes in single cells.

Laparoscopic repair of sliding inguinal hernia in female children.

PURPOSE: To evaluate the safety and feasibility of laparoscopic repair of sliding inguinal hernia in female children. METHODS: Laparoscopic percutaneous extraperitoneal closure (LPEC) was performed in 482 female inguinal hernia children between 2006 and 2015. Fourteen of these patients were associated with sliding inguinal hernia, and these 14 patients were enrolled and reviewed retrospectively. RESULTS: The mean age and the body weight at the operation was 9.6 months and 7.8 kg. Seven patients required the reduction of the ovary under general anesthesia. Laparoscopy, however, revealed that five patients had severe sliding of fallopian tube into the inguinal canal. One of these five patients received a simple LPEC, but developed the recurrence due to the low ligation of the hernia sac, and needed the second hernia repair under inguinal approach. Other four patients with fallopian tube sliding required the dissection of the fallopian tube and peritoneal repair, or the conversion to inguinal approach; therefore they had longer surgical time compared to those without fallopian tube sliding. CONCLUSION: LPEC is safe and feasible for the sliding inguinal hernia repair except the cases with fallopian tube sliding. Patients with fallopian tube sliding required additional procedure or conversion to inguinal approach.

Phosphorylation of Serine 402 Regulates RacGAP Protein Activity of FilGAP Protein.

FilGAP is a Rho GTPase-activating protein (GAP) that specifically regulates Rac. FilGAP is phosphorylated by ROCK, and this phosphorylation stimulates its RacGAP activity. However, it is unclear how phosphorylation regulates cellular functions and localization of FilGAP. We found that non-phosphorylatable FilGAP (ST/A) mutant is predominantly localized to the cytoskeleton along actin filaments and partially co-localized with vinculin around cell periphery, whereas phosphomimetic FilGAP (ST/D) mutant is diffusely cytoplasmic. Moreover, phosphorylated FilGAP detected by Phos-tag is also mainly localized in the cytoplasm. Of the six potential phosphorylation sites in FilGAP tested, only mutation of serine 402 to alanine (S402A) resulted in decreased cell spreading on fibronectin. FilGAP phosphorylated at Ser-402 is localized to the cytoplasm but not at the cytoskeleton. Although Ser-402 is highly phosphorylated in serum-starved quiescent cells, dephosphorylation of Ser-402 is accompanied with the cell spreading on fibronectin. Treatment of the cells expressing wild-type FilGAP with calyculin A, a Ser/Thr phosphatase inhibitor, suppressed cell spreading on fibronectin, whereas cells transfected with FilGAP S402A mutant were not affected by calyculin A. Expression of constitutively activate Arf6 Q67L mutant stimulated membrane blebbing activity of both non-phosphorylatable (ST/A) and phosphomimetic (ST/D) FilGAP mutants. Conversely, depletion of endogenous Arf6 suppressed membrane blebbing induced by FilGAP (ST/A) and (ST/D) mutants. Our study suggests that Arf6 and phosphorylation of FilGAP may regulate FilGAP, and phosphorylation of Ser-402 may play a role in the regulation of cell spreading on fibronectin.

Anterior mediastinal tracheostomy with a median mandibular splitting approach in a Larsen syndrome patient with posterior cervical arthrodesis.

Larsen syndrome is a rare congenital connective tissue disorder characterized by multiple joint dislocations. A novel anterior mediastinal tracheostomy with a median mandibular splitting approach is presented for the treatment of airway obstruction in a Larsen syndrome patient with posterior cervical arthrodesis.

A case of tracheal agenesis surviving without mechanical ventilation after external esophageal stenting.

Tracheal agenesis is a rare and usually lethal congenital malformation of the forgut. Although some infants can be resuscitated with an intra-esophageal intubation temporarily, long-term airway management is difficult because of the collapsing airway. We report a long-term survivor with tracheal agenesis in whom a Gortex external esophageal stent using radial traction sutures was applied to prevent the esophagus from collapsing. The patient was discharged from our hospital without mechanical ventilation or oxygen inhalation at 10 months of age. Our procedure has a potential to establish a long-term steady airway in patients with tracheal agenesis. The detail of the procedure is presented and the related literature is reviewed.

Lack of distal esophageal contractions is a key determinant of gastroesophageal reflux disease after repair of esophageal atresia.

BACKGROUND/PURPOSE: The objective of this study is to investigate the characteristics of esophageal motor activity responsible for the development of gastroesophageal reflux (GER) in patients with esophageal atresia (EA). METHODS: The subjects consisted of 29 patients with EA (1 month to 19 years). Computerized esophageal manometry was conducted to investigate esophageal contractions at swallow. A topographic esophageal manometric analysis was conducted in each subject, providing 3-dimensional displays to reveal the pressure continuum representing esophageal contractions. RESULTS: Significant contractions in the middle esophagus just below the anastomosis were absent in all subjects. Contractions in the distal esophagus were conspicuously absent in 17 subjects. Of these 17, 6 had already undergone fundoplication, and 9 had symptomatic GER requiring fundoplication subsequent to this study. The remaining 12 patients had contractions in the distal esophagus and did not require medical/surgical intervention. A lack of distal esophageal contractions was significantly correlated with the development of GER (P < .001). There was a significant difference in esophageal acid exposure between the 2 groups (median, 38% vs 4%, P < .001). CONCLUSION: Lack of distal esophageal contractions indicating an impaired clearing capacity is considered a potential key determinant of GER in patients with EA.

Anorectal sleeve micromanometry for the diagnosis of Hirschsprung's disease in newborns.

BACKGROUND/PURPOSE: An accurate diagnosis is mandatory for surgery in newborns with Hirschsprung's disease (HD). Acetylcholinesterase staining of rectal suction biopsy specimens is widely performed in the diagnosis of HD, but results are sometimes incorrect or atypical in newborns. We report the usefulness of our method of anorectal manometry using a specially designed sleeve microassembly for the diagnosis of neonatal HD. METHODS: Anorectal manometry was conducted without sedation in 41 newborns, aged 2 to 30 days (19 newborns were within the first week of life), with abdominal distension. A silastic assembly with a 2-cm-long sleeve sensor and 5 side holes arrayed along the sleeve was designed to reduce the effects of displacement of pressure sensors relative to the anal sphincter. Rectoanal inhibitory reflex (RAIR) was examined with rectal balloon distension. RESULTS: Thirty-two subjects who showed falls of anal sphincter pressure fulfilling the criteria for RAIR were diagnosed to be without HD. Nine patients without an appropriate RAIR were subsequently confirmed to have HD based on operative pathologic findings. Parameters of anal sphincter function did not differ significantly between the subjects with and without RAIR. CONCLUSIONS: An anorectal sleeve micromanometric technique is useful in the diagnostic workup of newborns suspected of having HD.