Is AI essential? Examining the need for deep learning in image-activated sorting of Saccharomyces cerevisiae.

Artificial intelligence (AI) has become a focal point across a multitude of societal sectors, with science not being an exception. Particularly in the life sciences, imaging flow cytometry has increasingly integrated AI for automated management and categorization of extensive cell image data. However, the necessity of AI over traditional classification methods when extending imaging flow cytometry to include cell sorting remains uncertain, primarily due to the time constraints between image acquisition and sorting actuation. AI-enabled image-activated cell sorting (IACS) methods remain substantially limited, even as recent advancements in IACS have found success while largely relying on traditional feature gating strategies. Here we assess the necessity of AI for image classification in IACS by contrasting the performance of feature gating, classical machine learning (ML), and deep learning (DL) with convolutional neural networks (CNNs) in the differentiation of Saccharomyces cerevisiae mutant images. We show that classical ML could only yield a 2.8-fold enhancement in target enrichment capability, albeit at the cost of a 13.7-fold increase in processing time. Conversely, a CNN could offer an 11.0-fold improvement in enrichment capability at an 11.5-fold increase in processing time. We further executed IACS on mixed mutant populations and quantified target strain enrichment via downstream DNA sequencing to substantiate the applicability of DL for the proposed study. Our findings validate the feasibility and value of employing DL in IACS for morphology-based genetic screening of S. cerevisiae, encouraging its incorporation in future advancements of similar technologies.

Prediction of ethanol fermentation under stressed conditions using yeast morphological data.

A high sugar concentration is used as a starting condition in alcoholic fermentation by budding yeast, which shows changes in intracellular state and cell morphology under conditions of high-sugar stress. In this study, we developed artificial intelligence (AI) models to predict ethanol yields in yeast fermentation cultures under conditions of high-sugar stress using cell morphological data. Our method involves the extraction of high-dimensional morphological data from phase contrast images using image processing software, and predicting ethanol yields by supervised machine learning. The neural network algorithm produced the best performance, with a coefficient of determination (R2) of 0.95, and could predict ethanol yields well even 60 min in the future. Morphological data from cells cultured in low-glucose medium could not be used for accurate prediction under conditions of high-glucose stress. Cells cultured in high-concentration glucose medium were similar in terms of morphology to cells cultured under high osmotic pressure. Feeding experiments revealed that morphological changes differed depending on the fermentation phase. By monitoring the morphology of yeast under stress, it was possible to understand the intracellular physiological conditions, suggesting that analysis of cell morphology can aid the management and stable production of desired biocommodities.

Intelligent sort-timing prediction for image-activated cell sorting.

Intelligent image-activated cell sorting (iIACS) has enabled high-throughput image-based sorting of single cells with artificial intelligence (AI) algorithms. This AI-on-a-chip technology combines fluorescence microscopy, AI-based image processing, sort-timing prediction, and cell sorting. Sort-timing prediction is particularly essential due to the latency on the order of milliseconds between image acquisition and sort actuation, during which image processing is performed. The long latency amplifies the effects of the fluctuations in the flow speed of cells, leading to fluctuation and uncertainty in the arrival time of cells at the sort point on the microfluidic chip. To compensate for this fluctuation, iIACS measures the flow speed of each cell upstream, predicts the arrival timing of the cell at the sort point, and activates the actuation of the cell sorter appropriately. Here, we propose and demonstrate a machine learning technique to increase the accuracy of the sort-timing prediction that would allow for the improvement of sort event rate, yield, and purity. Specifically, we trained an algorithm to predict the sort timing for morphologically heterogeneous budding yeast cells. The algorithm we developed used cell morphology, position, and flow speed as inputs for prediction and achieved 41.5% lower prediction error compared to the previously employed method based solely on flow speed. As a result, our technique would allow for an increase in the sort event rate of iIACS by a factor of ~2.

AI-based forecasting of ethanol fermentation using yeast morphological data.

Several industries require getting information of products as soon as possible during fermentation. However, the trade-off between sensing speed and data quantity presents challenges for forecasting fermentation product yields. In this study, we tried to develop AI models to forecast ethanol yields in yeast fermentation cultures, using cell morphological data. Our platform involves the quick acquisition of yeast morphological images using a nonstaining protocol, extraction of high-dimensional morphological data using image processing software, and forecasting of ethanol yields via supervised machine learning. We found that the neural network algorithm produced the best performance, which had a coefficient of determination of >0.9 even at 30 and 60 min in the future. The model was validated using test data collected using the CalMorph-PC(10) system, which enables rapid image acquisition within 10 min. AI-based forecasting of product yields based on cell morphology will facilitate the management and stable production of desired biocommodities.

Are droplets really suitable for single-cell analysis? A case study on yeast in droplets.

Single-cell analysis has become one of the main cornerstones of biotechnology, inspiring the advent of various microfluidic compartments for cell cultivation such as microwells, microtrappers, microcapillaries, and droplets. A fundamental assumption for using such microfluidic compartments is that unintended stress or harm to cells derived from the microenvironments is insignificant, which is a crucial condition for carrying out unbiased single-cell studies. Despite the significance of this assumption, simple viability or growth tests have overwhelmingly been the assay of choice for evaluating culture conditions while empirical studies on the sub-lethal effect on cellular functions have been insufficient in many cases. In this work, we assessed the effect of culturing cells in droplets on the cellular function using yeast morphology as an indicator. Quantitative morphological analysis using CalMorph, an image-analysis program, demonstrated that cells cultured in flasks, large droplets, and small droplets significantly differed morphologically. From these differences, we identified that the cell cycle was delayed in droplets during the G1 phase and during the process of bud growth likely due to the checkpoint mechanism and impaired mitochondrial function, respectively. Furthermore, comparing small and large droplets, cells cultured in large droplets were morphologically more similar to those cultured in a flask, highlighting the advantage of increasing the droplet size. These results highlight a potential source of bias in cell analysis using droplets and reinforce the significance of assessing culture conditions of microfluidic cultivation methods for specific study cases.

Characterization of lysine acetylation in the peripheral subunit-binding domain of the E2 subunit of the pyruvate dehydrogenase-2-oxoglutarate dehydrogenase hybrid complex from Corynebacterium glutamicum.

In Corynebacterium glutamicum, pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (ODH) form a unique hybrid complex in which CgE1p and CgE1o are associated with the CgE2-CgE3 subcomplex. We analyzed the role of a lysine acetylation site in the peripheral subunit-binding domain of CgE2 in PDH and ODH functions. Acetylation-mimic substitution at Lys391 of CgE2 severely reduced the interaction of CgE2 with CgE1p and CgE3, but not with CgE1o, indicating the critical role of this residue in the assembly of CgE1p and CgE3 into the complex. It also suggested that Lys391 acetylation inhibited the binding of CgE1p and CgE3 to CgE2, thereby affecting PDH and ODH activities. Interestingly, the CgE2-K391R variant strain showed increased l-glutamate production and reduced pyruvate accumulation. Kinetic analysis suggested that the increased affinity of the K391R variant toward pyruvate might be advantageous for l-glutamate production.

[A Patient with Rectal Cancer Who Has Been Associated with Long-Term Complete Response to Chemotherapy].

A female in her forties was diagnosed with rectal cancer involving the uterus and bladder and with multiple lymph node metastasis. Chemotherapy was successful and resulted in complete clinical response 1 year and 2 months after commencement. She continues chemotherapy without resection of the primary tumor. The complete response has been maintained for 8 years and 11 months.

In vitro reconstitution and characterization of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase hybrid complex from Corynebacterium glutamicum.

Pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (ODH) are critical enzymes in central carbon metabolism. In Corynebacterium glutamicum, an unusual hybrid complex consisting of CgE1p (thiamine diphosphate-dependent pyruvate dehydrogenase, AceE), CgE2 (dihydrolipoamide acetyltransferase, AceF), CgE3 (dihydrolipoamide dehydrogenase, Lpd), and CgE1o (thiamine diphosphate-dependent 2-oxoglutarate dehydrogenase, OdhA) has been suggested. Here, we elucidated that the PDH-ODH hybrid complex in C. glutamicum probably consists of six copies of CgE2 in its core, which is rather compact compared with PDH and ODH in other microorganisms that have twenty-four copies of E2. We found that CgE2 formed a stable complex with CgE3 (CgE2-E3 subcomplex) in vitro, hypothetically comprised of two CgE2 trimers and four CgE3 dimers. We also found that CgE1o exists mainly as a hexamer in solution and is ready to form an active ODH complex when mixed with the CgE2-E3 subcomplex. Our in vitro reconstituted system showed CgE1p- and CgE1o-dependent inhibition of ODH and PDH, respectively, actively supporting the formation of the hybrid complex, in which both CgE1p and CgE1o associate with a single CgE2-E3. In gel filtration chromatography, all the subunits of CgODH were eluted in the same fraction, whereas CgE1p was eluted separately from CgE2-E3, suggesting a weak association of CgE1p with CgE2 compared with that of CgE1o. This study revealed the unique molecular architecture of the hybrid complex from C. glutamicum and the compact-sized complex would provide an advantage to determine the whole structure of the unusual hybrid complex.

Fournier's Gangrene in a Patient with Type 2 Diabetes Mellitus Treated with Empagliflozin: A Case Report.

Sodium-glucose cotransporter-2 (SGLT2) inhibitors have been reported as possibly associated with Fournier's gangrene (FG). This case report describes a 34-year-old Japanese man who was diagnosed with FG after the administration of empagliflozin for type 2 diabetes mellitus (T2DM). He presented with pain and swelling in the perineum and groin 142 days after initiating empagliflozin. The clinical features, laboratory data, and computed tomographic findings were consistent with FG. Surgical drainage and debridement of necrotic tissues were performed immediately after admission to our hospital. The patient had no complications of diabetes before the onset of FG. Glycemic management was good at the time of FG onset. This case suggests a possible association between empagliflozin and FG. We report a case of FG in a patient during a period of good glycemic management following treatment with empagliflozin. We recommend further awareness of this relationship and suggest the need for additional research.

Dynamic changes in lysine acetylation and succinylation of the elongation factor Tu in Bacillus subtilis.

Nε-lysine acetylation and succinylation are ubiquitous post-translational modifications in eukaryotes and bacteria. In the present study, we showed a dynamic change in acetylation and succinylation of TufA, the translation elongation factor Tu, from Bacillus subtilis. Increased acetylation of TufA was observed during the exponential growth phase in LB and minimal glucose conditions, and its acetylation level decreased upon entering the stationary phase, while its succinylation increased during the late stationary phase. TufA was also succinylated during vegetative growth under minimal citrate or succinate conditions. Mutational analysis showed that triple succinylation mimic mutations at Lys306, Lys308 and Lys316 in domain-3 of TufA had a negative effect on B. subtilis growth, whereas the non-acylation mimic mutations at these three lysine residues did not. Consistent with the growth phenotypes, the triple succinylation mimic mutant showed 67 % decreased translation activity in vitro, suggesting a possibility that succinylation at the lysine residues in domain-3 decreases the translation activity. TufA, including Lys308, was non-enzymatically succinylated by physiological concentrations of succinyl-CoA. Lys42 in the G-domain was identified as the most frequently modified acetylation site, though its acetylation was likely dispensable for TufA translation activity and growth. Determination of the intracellular levels of acetylating substrates and TufA acetylation revealed that acetyl phosphate was responsible for acetylation at several lysine sites of TufA, but not for Lys42 acetylation. It was speculated that acetyl-CoA was likely responsible for Lys42 acetylation, though AcuA acetyltransferase was not involved. Zn2+-dependent AcuC and NAD+-dependent SrtN deacetylases were responsible for deacetylation of TufA, including Lys42. These findings suggest the potential regulatory roles of acetylation and succinylation in controlling TufA function and translation in response to nutrient environments in B. subtilis.

A case of spinal myoclonus in a patient with elective cesarean section.

BACKGROUND: Transient myoclonic involuntary movements, typically referred to as spinal myoclonus (SM), rarely develop in the extremities following neuraxial anesthesia (NA). NA indications in patients with history of SM following NA (SM-NA) are unknown. CASE PRESENTATION: A 33-year-old woman developed SM-NA after elective cesarean section (CS). Approximately 130 min after spinal anesthesia induction, she began exhibiting involuntary movements, which became most severe after approximately 3 h. The involuntary movements gradually decreased without treatments and disappeared after approximately 5 h. The patient underwent CS on three occasions. The first CS (age, 29 years) was under a combination of spinal and epidural anesthesia. The third CS (age, 35 years) was completed using only spinal anesthesia. There were no neurological events during the postoperative courses for the first and third CS. CONCLUSIONS: SM-NA can unexpectedly occur, and history of SM-NA may not be contraindicative for repeated NA.

[Resected Pulmonary Cryptococcosis Suspected as Lung Cancer;Report of a Case].

We report a case of pulmonary cryptococcosis suspected of lung cancer. A 65-year-old woman had an abnormal shadow on chest X-ray. A solitary nodule, 12 mm in size, with pleural indentation and spicula in S3 of the left lung was found on chest computed tomography. A serum cryptococcal antigen was negative. An abnormal accumulation of fluoro-2-deoxy-D-glucose(FDG)in the nodule was found with the standardized uptake value (SUV) max 5.04, suggesting lung cancer. The nodule was diagnosed as pulmonary cryptococcosis by surgical resection.

Severe Dextran-Induced Anaphylactic Shock during Induction of Hypertension-Hypervolemia-Hemodilution Therapy following Subarachnoid Hemorrhage.

Dextran is a colloid effective for volume expansion; however, a possible side effect of its use is anaphylaxis. Dextran-induced anaphylactoid reaction (DIAR) is a rare but severe complication, with a small dose of dextran solution sufficient to induce anaphylaxis. An 86-year-old female who underwent clipping for a ruptured cerebral aneurysm was admitted to the intensive care unit. Prophylactic hypertension-hypervolemia-hemodilution therapy was induced for cerebral vasospasm following a subarachnoid hemorrhage. The patient went into severe shock after administration of dextran for volume expansion, and dextran administration was immediately discontinued. The volume administered at that time was only 0.8 mL at the most. After fluid resuscitation with a crystalloid solution, circulatory status began to recover. However, cerebral vasospasm occurred and the patient's neurological condition deteriorated. Five weeks after the shock, she was diagnosed with hypersensitivity to dextran by a skin test. When severe hypotension occurs after dextran administration, appropriate treatments for shock should be performed immediately with discontinuation of dextran solution. Although colloid administration is recommended in some guidelines and researches, it is necessary to consider concerning the indication for volume expansion as well as the risk of colloid administration.

Practical management and treatment of pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (NETs) are uncommon disease, about which little is known. Pancreatic NETs are usually slow growing and their malignant potential are often underestimated. The management of this disease poses a challenge because of the heterogeneous clinical presentation and varying degrees of aggressiveness. Recently, several guidelines for the management of pancreatic NETs have been established and help to devise clinical strategy. In the treatment algorithms, however, a lot of uncertain points are included. Practical treatment decisions of pancreatic NETs are still sometimes made in a patient- and/or physicians-oriented manner. The tumor grading system proposed by the European Neuroendocrine Tumor Society (ENETS) gives important prognostic information, however, the implication of grading regarding medical treatment strategies to choose has not yet been clarified. Moreover, the place of surgical treatment is unclear in the overall management course of advanced pancreatic NETs. In some cases, practical management and treatment have to be individualized depending on predominant symptoms, tumor spread, and general health of the patients. Current issues and a few points to make a strategy in the management of pancreatic NETs would be reviewed.

Rapid determination of isoamyl nitrite in pharmaceutical preparations by flow injection analysis with on-line UV irradiation and luminol chemiluminescence detection.

Isoamyl nitrite is used as a therapeutic reagent for cardiac angina and as an antidote for cyanide poisoning, but it is abused because of its euphoric properties. Therefore, a method to determine isoamyl nitrite is required in many fields, including pharmaceutical and forensic studies. In this study, a simple, rapid and sensitive method for the determination of isoamyl nitrite was developed using a flow injection analysis system equipped with a chemiluminescence detector and on-line photoreactor. This method is based on on-line ultraviolet irradiation of isoamyl nitrite and subsequent luminol chemiluminescence detection without the addition of an oxidant. A linear standard curve was obtained up to 1.0 µM of isoamyl nitrite with a detection limit (blank + 3SD) of 0.03 µM. The method was successfully applied to determine isoamyl nitrite content in pharmaceutical preparations.

[Complete response in a case of anastomotic recurrence of rectal cancer treated with S-1 monotherapy].

A 63-year-old woman underwent a low anterior resection for rectal cancer in 2002.A n anastomotic recurrence was diagnosed in July 2011.S he rejected the possibility of colostomy as radical surgery.Chemotherapy consisting of capecitabine+ oxaliplatin (XELOX) or folinic acid, fluorouracil, and oxaliplatin (FOLFOX6) + bevacizumab were not possible because of high costs. In view of the lower costs and the potential for ambulation, S-1 monotherapy was started. After 3 months, a reduction in the recurrent lesion was observed.After 19 months, the recurrent lesion revealed a scar, which was judged by biopsy to be Group 1.We had achieved a pathological complete response (CR).The standard treatment for recurrent colon cancer is surgical resection or multidrug chemotherapy. However, in view of a patient's quality of life (QOL), S-1 monotherapy may be considered as a potential therapy.

[No recurrent case of the combination of surgical management with chemotherapy].

A 61-year-old man consulted a nearby doctor with the chief complaint of lassitude in June 2010. Blood tests revealed a hemoglobin level of 3.7 g/dL. The observation of significant anemia resulted in a full medical workup. On computed tomography (CT) findings, a large mass (17 × 10 cm in diameter) was found in the abdominal region. The lumen was distended with accumulation of air and fluid. In addition, I continued with a bladder wall, but did not recognize the abnormality that was apparent to a bladder lumen. There was no evidence of ileus. A diagnosis of appendix cancer or sarcoma was made preoperatively. During surgery, the bladder was observed to have some permeation, but the bladder wall contained a lesion of small intestinal origin with only slight permeation. The neighboring small intestine was surrounded by the tumor, with 3 sites of penetration. Histopathologic diagnosis indicated a high-risk gastrointestinal stromal tumor (GIST) with mitotic figures (44/50 high power fields). In accordance with the recommended guidelines, imatinib was administered for 1 year. Two sites of recurrences were observed by CT after discontinuing imatinib. A second operation was performed without increase because of the absence other lesions. Separate lesions in the sigmoid colon and jejunum were removed surgically. The tumor in the sigmoid colon was a lesion with high denaturation for mesenchymal system tumor such as GISTs by pathologic diagnosis. The lesion in the small mesentery was a suture granuloma. In this case, the combination of surgical management with chemotherapy resulted in good quality of life with no recurrence despite the presence of a high-risk GIST.

Role of resection of the primary pancreatic neuroendocrine tumor in the multidisciplinary treatment of patients with unresectable synchronous liver metastases: a case series.

CONTEXT: Liver metastases have often existed in patients who have pancreatic neuroendocrine tumors (pNETs) at the time of diagnosis. In the management of patients of pNETs with unresectable liver metastases, the clinical efficacy of surgery to primary pancreatic tumor has been controversial. We presented four patients who were treated with resection of primary pancreatic tumor, trans-arterial hepatic treatment and systemic therapies. We reviewed literatures and discussed about role of resection of primary pancreatic tumor in the multidisciplinary treatment. METHODS: We retrieved medical records of patients who had been histopathologically diagnosed as pNETs at our institution between April 2000 and March 2006, and found 4 patients who had pNETs with unresectable synchronous liver metastases and no extrahepatic metastases. All patients received resection of primary tumor. Patients' demographics, pathology, treatment, short- and long-term outcome were examined. RESULTS: In short-term outcome analysis, delayed gastric emptying was developed in one patient who received pancreaticoduodenectomy. There were no other significant postoperative complications. As for long-term outcome, two patients who received distal pancreatectomy, sequential trans-arterial treatments and systemic therapies could survive for long time relatively. They died 92 and 73 months after the first treatment, respectively. One patient who received distal pancreatectomy and trans-arterial treatment died from unrelated disease 14 months after the first treatment. Another patient who received preoperative trans-arterial treatments and pancreaticoduodenectomy rejected postoperative trans-arterial treatment, was treated with systemic therapies and died 37 months after the initial treatment. CONCLUSIONS: Resection of primary pNETs would be considered as an optional treatment for the selected patients who had unresectable synchronous liver metastases in the process of the multidisciplinary approach.

Late recurrence after resection of mass-forming intrahepatic cholangiocarcinoma: report of a case.

The outcome after surgical resection for intrahepatic cholangiocarcinoma has not been satisfactorily evaluated due to its malignant behavior. Surgical resection, however, has the potential to improve the prognosis and may allow surgeons to experience rare cases with long survival. This report presents the case of a patient who developed recurrence 9 years after resection of intrahepatic cholangiocarcinoma. A 76-year-old female was diagnosed to have intrahepatic cholangiocarcinoma and underwent an extended right posterior subsegmentectomy. The gross appearance showed a mass-forming type tumor. The histopathological examination revealed well to moderately differentiated adenocarcinoma associated with portal vein invasion. Subcutaneous metastasis in the head as the first sign of relapse was diagnosed 9 years after hepatectomy. The histopathological findings of the subcutaneous tumor were similar to those of the intrahepatic cholangiocarcinoma, thus suggesting metastasis from intrahepatic cholangiocarcinoma. Positron emission tomography with 2-[fluorine-18]-fluoro-2-deoxy-D-glucose was useful for detecting multiple metastases. Long-term follow-up for more than 5 years is recommended because the present case shows that late recurrence of intrahepatic cholangiocarcinoma occurs even 5 years after resection.