Mixotrophic culture enhances fucoxanthin production in the haptophyte Pavlova gyrans.

Fucoxanthin is a versatile substance in the food and pharmaceutical industries owing to its excellent antioxidant and anti-obesity properties. Several microalgae, including the haptophyte Pavlova spp., can produce fucoxanthin and are potential industrial fucoxanthin producers, as they lack rigid cell walls, which facilitates fucoxanthin extraction. However, the commercial application of Pavlova spp. is limited owing to insufficient biomass production. In this study, we aimed to develop a mixotrophic cultivation method to increase biomass and fucoxanthin production in Pavlova gyrans OPMS 30543X. The effects of culturing OPMS 30543X with different organic carbon sources, glycerol concentrations, mixed-nutrient conditions, and light intensities on the consumption of organic carbon sources, biomass production, and fucoxanthin accumulation were analyzed. Several organic carbon sources, such as glycerol, glucose, sucrose, and acetate, were examined, revealing that glycerol was well-consumed by the microalgae. Biomass and fucoxanthin production by OPMS 30543X increased in the presence of 10 mM glycerol compared to that observed without glycerol. Metabolomic analysis revealed higher levels of the metabolites related to the glycolytic, Calvin-Benson-Bassham, and tricarboxylic acid cycles under mixotrophic conditions than under autotrophic conditions. Cultures grown under mixotrophic conditions with a light intensity of 100 µmol photons m-2 s-1 produced more fucoxanthin than autotrophic cultures. Notably, the amount of fucoxanthin produced (18.9 mg/L) was the highest reported thus far for Pavlova species. In conclusion, the use of mixotrophic culture is a promising strategy for increasing fucoxanthin production in Pavlova species. KEY POINTS: • Glycerol enhances biomass and fucoxanthin production in Pavlova gyrans • Metabolite levels increase under mixotrophic conditions • Mixotrophic conditions and medium-light intensity are appropriate for P. gyrans.

Clinical impact of fluoropolymer-based drug-eluting stent thrombosis in femoropopliteal artery occlusive lesions.

BACKGROUND: Endovascular treatment (EVT) is recommended for superficial femoral artery (SFA) lesions, and good results have been reported after implantation of drug-eluting stents (DES) for SFA. However, the major concern after implantation is acute thrombosis during the follow-up period, resulting in major amputation and major adverse limb events. In this study, we examined the incidence and outcome of acute thrombosis after DES implantation in the SFA. OBJECTIVES AND METHODS: DES implantation for a femoropopliteal lesion was performed in 288 patients at multiple centers in Japan from 2019 to 2021. A total of 25 patients (8.6%) with DES acute occlusion were analyzed retrospectively. The primary endpoint was amputation-free survival (AFS) after acute occlusion. RESULTS: The median patient age was 77 years, with 48% having diabetes, 40% undergoing maintenance dialysis, and 66% having chronic limb-threatening ischemia (CLTI). The mean time from initial DES implantation to acute occlusion was 153.5 ± 177.6 days, with a median of 104 days. EVT was performed in 18 patients (72%), surgical revascularization in 3 (12%), and conservative treatment in 4 (16%). Two deaths within 30 days were both due to sepsis. No major amputation or major adverse cardiovascular events occurred within 30 days. The 1-year rates of patency and freedom from target lesion revascularization after DES thrombosis were 22.9% and 48.8%, respectively. AFS at 1 year was 55.1%. CONCLUSION: Acute DES occlusion is relatively frequent, and the outcome is poor. Therefore, the indication of DES implantation for a complex SFA lesion may require careful consideration. Further investigation may be needed in DES implantation for a complex SFA lesion.

Cerebral activation caused by dental sounds: a functional magnetic resonance imaging study.

Dental drilling sounds can induce anxiety in some patients. This study aimed to use functional magnetic resonance imaging (fMRI) to assess the relationship between dental fear and auditory stimuli. Thirty-four right-handed individuals (21 women and 13 men; average age, 31.2 years) were selected. The level of dental fear was assessed using the dental fear survey (DFS). Based on a threshold DFS score > 52, participants were categorized into two groups: dental fear (DF) group (n = 12) and control group (n = 22). Two types of stimuli were presented in a single session: dental and neutral sounds. Cerebral activation during the presentation of these sounds was evaluated using contrast-enhanced blood oxygenation level-dependent fMRI. In the DF group, dental sounds induced significantly stronger activation in the left inferior frontal gyrus and left caudate nucleus (one-sample t test, P < 0.001). In contrast, in the control group, significantly stronger activation was observed in the bilateral Heschl's gyri and left middle frontal gyrus (one-sample t test, P < 0.001). Additionally, a two-sample t test revealed that dental sounds induced a significantly stronger activation in the left caudate nucleus in the DF group than in the control group (P < 0.005). These findings suggest that the cerebral activation pattern in individuals with DF differs from that in controls. Increased activation of subcortical regions may be associated with sound memory during dental treatment.

Circular cell culture for sustainable food production using recombinant lactate-assimilating cyanobacteria that supplies pyruvate and amino acids.

Recently, we reported a circular cell culture (CCC) system using microalgae and animal muscle cells for sustainable culture food production. However, lactate accumulation excreted by animal cells in the system characterized by medium reuse was a huge problem. To solve the problem, as an advanced CCC, we used a lactate-assimilating cyanobacterium Synechococcus sp. PCC 7002, using gene-recombination technology that synthesises pyruvate from lactate. We found that the cyanobacteria and animal cells mutually exchanged substances via their waste media: (i) cyanobacteria used lactate and ammonia excreted by animal muscle cells, and (ii) the animal cells used pyruvate and some amino acids excreted by the cyanobacteria. Because of this, animal muscle C2C12 cells were amplified efficiently without animal serum in cyanobacterial culture waste medium in two cycles (first cycle: 3.6-fold; second cycle: 3.9-fold/three days-cultivation) using the same reuse medium. We believe that this advanced CCC system will solve the problem of lactate accumulation in cell culture and lead to efficient cultured food production.

Evaluation of antero-posterior instability of the hip using modified Lequesne's false profile view.

BACKGROUND: Accurate evaluation of hip instability is critical for the diagnosis and successful treatment of developmental dysplasia of the hip (DDH). However, dynamic evaluation of hip instability is not well established. This study aimed to use the lateral view from a radiograph to evaluate dynamic antero-posterior hip instability in patients with DDH. METHODS: Seventy-four patients (92 hips) with DDH (DDH group) and 46 patients (59 hips) without hip pain and DDH (Control group) were examined. A false profile view (FPV) according to Lequesne was taken at standard and 90° flexion with the hip of interest defined as functional FPV; the translation of the center of the femoral head (CFH) obtained from the functional FPV was measured. As a validation test, we measured the anterior translation of the CFH using ultrasonography (US). RESULTS: There was a significant difference between the two groups in the translation of the CFH (p < 0.01). The degree of CFH translation depended on the severity of DDH (lateral center edge angle, r = -0.56, p < 0.01; vertical center anterior margin angle, r = -0.57, p<0.01) and lateralization of the femoral head (head lateralization index, r = 0.54, p < 0.01). There was a significant correlation between functional FPV and US measurements (r = 0.71, p < 0.01). CONCLUSION: The present study confirmed that antero-posterior hip instability in DDH patients can be detected using functional FPV. Our novel measurement, as a new method for assessing hip instability, may be useful for evaluating hip dynamic instability in diagnosing the etiology, and determining and evaluating the treatment for DDH at lower cost and improved accessibility.

Relationships among hip instability, iliofemoral ligament, and pain in patients with developmental dysplasia of the hip.

BACKGROUND: To evaluate the relationships among hip instability, pain, and morphology of the iliofemoral ligament (ILFL) in patients with developmental dysplasia of the hip (DDH) using ultrasonography (US). METHODS: We reviewed 86 patients (109 hips) with DDH (Group D), 40 patients (46 hips) with borderline hip dysplasia (BDDH) (Group B) and 20 patients (23 hips) without hip pain and bony abnormality (control group). Group D was classified into three subgroups-the severe (group SP), moderate (group MP), and none/mild (group NMP) hip pain groups-using the visual analogue scale (VAS). For evaluating hip instability and ILFL morphology, the distance between the anterior edge of the anterior inferior iliac spine (AIIS) and the horizontal line to the femoral head, and ILFL thickness were measured using US. The difference between the distance in the neutral position and Patrick position was calculated and defined as the femoral head translation distance (FTD). RESULTS: FTD and ILFL thickness in group D were significantly larger than those in the control group and group B (P < 0.05). There was a significant positive correlation between FTD and ILFL thickness in three groups (r = 0.57, P < 0.05; r = 0.55, P < 0.05; r = 0.62, P < 0.05, respectively). FTD and ILFL thickness in group SP were significantly larger than those in group NMP (P < 0.05). FTD and ILFL thickness in group D had significantly negative correlations with the lateral center edge (r = -0.54, P < 0.05; r = -0.40, P < 0.05, respectively) and vertical-center-anterior angle (r = -0.51, P < 0.05; r = -0.43, P < 0.05, respectively). CONCLUSIONS: Acetabular bony deficiency, especially in the anterior and lateral region can result in antero-posterior hip instability, leading to thickened ILFL and hip pain, even in patients with BDDH. These findings may facilitate our understanding and treatment of patients with DDH. When hip instability is suspected, hip US examination may help confirm the diagnosis and assist in providing objective clinical diagnostic evidence.

Investigating the subchondral trabecular bone microstructure in patients with osteonecrosis of the femoral head using multi-detector row computed tomography.

OBJECTIVES: To analyse the microstructural changes of subchondral trabecular bone in patients with osteonecrosis of the femoral head (ONFH) using multi-detector row computed tomography (MDCT). METHODS: We retrospectively investigated 76 hips in 50 patients diagnosed with ONFH between 2017 and 2021. Groups 1, 2, 3, and 4 comprised hips without ONFH, ONFH without femoral head collapse (FHC), ONFH with mild collapse (<2 mm), and ONFH with severe collapse (>2 mm), respectively. All patients underwent MDCT, and the subchondral trabecular bone microstructure was assessed. Regions of interests were set at the lateral boundary of the femoral head necrotic lesion and centre of the acetabular weight-bearing portion. RESULTS: In both the femoral head and the acetabular regions, there were significant differences in Groups 2 and 3 compared to Group 1, with increased volumetric bone mineral density and apparent bone volume fraction, and more plate-like with increased connectivity, indicating that osteosclerotic changes were occurring. CONCLUSIONS: In both the femoral head and the acetabular regions, osteosclerotic changes of subchondral trabecular bone microstructure were present before FHC.

Pulmonary hypertension with bronchopulmonary dysplasia: Aichi cohort study.

BACKGROUND: The incidence of pulmonary hypertension (PH) associated with bronchopulmonary dysplasia (BPD) has not been investigated in regional cohorts. The aim of this study was to clarify the incidence of PH associated with BPD in all very low birthweight infants (VLBWIs) born during the study period in Aichi Prefecture, Japan. METHODS: We conducted a retrospective observational cohort study of all VLBWIs born in Aichi Prefecture. The inclusion criteria were VLB, birth between 1 January 2015 and 31 December 2015, and admission to any neonatal intensive care unit in Aichi Prefecture. BPD28d and BPD36w were defined as the need for supplemental oxygen or any respiratory support at 28 days of age or 36 weeks of postmenstrual age (PMA). The primary outcome was the incidence of PH after 36 weeks' PMA (PH36w) in VLBWIs with BPD28d and BPD36w. The secondary outcomes were the clinical factors related to PH36w in BPD36w patients. Mann-Whitney U-test and Fisher's exact test were used for univariate analysis. Differences were considered statistically significant at P < 0.05. Risk ratio (RR) and 95% confidence interval (CI) were also evaluated. RESULTS: A total of 441 patients were analyzed. A total of 217 and 131 patients met the definition of BPD28d and BPD36w, respectively. Nine patients were diagnosed with PH36w (4.2% and 6.9% of the BPD28d and BPD36w patients, respectively). The presence of oligohydramnios (RR, 2.71; 95% CI: 1.55-4.73, P = 0.014) and sepsis (RR, 3.62; 95% CI: 1.51-8.63, P = 0.025) was significant in the PH36w patients. CONCLUSIONS: The incidence of PH36w was 4.2% and 6.9% in the BPD28d and BPD36w patients, respectively. Oligohydramnios and sepsis were significantly associated with PH36w in VLBWIs.

Rapidly Progressive Stenosis of the Left Main Trunk Ostium Starting 21 Months After Stent Implantation.

Rapidly progressive in-stent restenosis (ISR) after stent deployment from the left main trunk (LMT) to the left anterior descending artery (LAD) without plaque at the LMT ostium has not been reported. A 60-year-old Japanese man with a history of scleroderma, pulmonary fibrosis, and type 2 diabetes developed acute myocardial infarction of the right coronary artery (RCA) and was treated by emergency percutaneous coronary intervention (PCI) for RCA. Nine days later he underwent PCI from the LMT to the LAD. Follow-up coronary angiography (CAG) at 9 and 21 months post-PCI did not reveal ISR in any lesion, but the patient experienced cardiac arrest at 25 months post-PCI. Emergency CAG after resuscitation revealed ISR of the LMT ostium; emergency PCI was conducted. The development of ISR at the ostium of the LMT although the patient was free of plaque 4 months before is extremely unusual. This rare ISR of the LMT ostium progressed rapidly after follow-up CAG revealed no ISR at 21 months post-stent implantation.

Metabolic Engineering for Carotenoid Production Using Eukaryotic Microalgae and Prokaryotic Cyanobacteria.

Eukaryotic microalgae and prokaryotic cyanobacteria are diverse photosynthetic organisms that produce various useful compounds. Due to their rapid growth and efficient biomass production from carbon dioxide and solar energy, microalgae and cyanobacteria are expected to become cost-effective, sustainable bioresources in the future. These organisms also abundantly produce various carotenoids, but further improvement in carotenoid productivity is needed for a successful commercialization. Metabolic engineering via genetic manipulation and mutational breeding is a powerful tool for generating carotenoid-rich strains. This chapter focuses on carotenoid production in microalgae and cyanobacteria, as well as strategies and potential target genes for metabolic engineering. Recent achievements in metabolic engineering that improved carotenoid production in microalgae and cyanobacteria are also reviewed.

Supramolecular Chemistry-Based One-Pot High-Efficiency Separation of Solubilizer-Free Pure Semiconducting Single-Walled Carbon Nanotubes: Molecular Strategy and Mechanism.

Single-walled carbon nanotubes (SWCNTs) have the potential to revolutionize nanoscale electronics and power sources; however, their low purity and high separation cost limit their use in practical applications. Here we present a supramolecular chemistry-based one-pot, less expensive, scalable, and highly efficient separation of a solubilizer/adsorbent-free pure semiconducting SWCNT (sc-SWCNT) using flavin/isoalloxazine analogues with different substituents. On the basis of both experimental and computational simulations (DFT study), we have revealed the molecular requirements of the solubilizers as well as provided a possible mechanism for such a highly efficient selective sc-SWCNT separation. The present sorting method is very simple (one-pot) and gives a promising sc-SWCNT separation methodology. Thus, the study provides insight for the molecular design of an sc-SWCNT solubilizer with a high (n,m)-chiral selectivity, which benefits many areas including semiconducting nanoelectronics, thermoelectric, bio and energy materials, and devices using solubilizer-free very pure sc-SWCNTs.

Competition between Conversion Reaction with Cerium Dioxide and Lithium Plating in Superconcentrated Electrolyte.

Li-ion insertion into cerium dioxide (CeO2) and its subsequent conversion reaction were studied using a CeO2/copper composite electrode in a superconcentrated electrolyte of lithium bis(fluorosulfonyl)amide (LiFSA) and methylphenylamino-di(trifluoroethyl) phosphate (PNMePh) under conditions promoting Li plating/stripping. Since the conversion reaction potential with CeO2 generally lies above the Li plating/stripping level, the conversion ideally occurs first in the cathodic scan. However, the conversion reaction was delayed until after the Li plating in the superconcentrated electrolyte contrary to expectations, whereas this phenomenon was unobserved in a dilute LiFSA/PNMePh electrolyte. Energy-dispersive X-ray spectroscopy and electrochemical impedance analysis indicated that the reversed order of the electrochemical behaviors was caused by the solid electrolyte interphase (SEI) on the CeO2, which had a different material composition and a higher interfacial resistance than the SEI on electrodeposited metallic lithium.

Fe2+ Ions Alleviate the Symptom of Citrus Greening Disease.

Citrus greening (CG) is among the most devastating citrus diseases worldwide. CG-infected trees exhibit interveinal chlorotic leaves due to iron (Fe) deficiency derived from CG; thus, Fe content is lower in infected leaves than in healthy leaves. In this study, we demonstrated that the foliar application of Fe2+ relieves the symptom of CG infection in citrus trees. We applied Fe2+ and citrate to the leaves of infected rough lemon plants. Following this treatment, a reduction in the number of yellow symptomatic leaves was observed, and their growth was restored. Using chlorophyll content as an index, we screened for effective Fe complexes and found that a high ratio of citrate to Fe2+ in the applied solution led to effects against CG in Shikuwasa trees. A high proportion of Fe2+ to total Fe was another key factor explaining the effectiveness of the solution in CG infection, indicating the importance of Fe2+ absorption into plant cells. We confirmed the proportion of Fe2+ to total Fe through the high correlation of reflectometry data via a triazine reaction and X-ray absorption fine structure analysis. These results demonstrate that the foliar application of a high-Fe2+ citrate solution can restore the growth of CG diseased trees.

High-Dose Chemotherapy with Autologous Stem Cell Transplantation in Primary Central Nervous System Lymphoma: Data From the Japan Society for Hematopoietic Cell Transplantation Registry.

High-dose chemotherapy (HDT) with autologous stem cell transplantation (ASCT) has been shown to improve the prognosis of patients with central nervous system (CNS) lymphoma. We queried the Japan Society for Hematopoietic Cell Transplantation Registry for 2006 to 2015 to analyze the outcomes of 102 patients with primary CNS lymphoma (PCNSL) who underwent first HDT/ASCT. The median patient age was 54 years (range, 20 to 74 years), and 65 patients were treated in an upfront setting. With a median duration of follow-up of 44 months, the 5-year overall survival (OS) and progession-free survival (PFS) were 54.9% and 38.4%, respectively. There were no significant differences in OS and PFS between upfront and salvage HDT/ASCT. Because thiotepa, a key agent in HDT/ASCT for PCNSL, has been unavailable since 2011 in Japan, the HDT regimens used were not uniform. Thiotepa-containing HDT was received by 16 out of 32 patients before 2010, but by only 2 of 70 patients after 2011. Thiotepa-containing HDT was associated with better PFS (P = .019), lower relapse (P = .042), and a trend toward a survival benefit. In multivariate analysis, noncomplete remission at HDT/ASCT was an independent predictor for OS (hazard ratio [HR], 2.40; 95% confidence interval [CI], 1.25 to 4.58; P = .008) and thiotepa-containing HDT remained significant for PFS (HR, .42; 95% CI, .19 to .95; P = .038). These results confirm the activity of thiotepa-containing regimens.

Factors related to survival discharge in trisomy 18: A retrospective multicenter study.

Infants with trisomy 18 (T18) previously had a poor prognosis; however, the intensive care of these patients has markedly diversified the prognosis. We investigated the current situation of patients with T18, clarified factors for survival discharge, and surveyed actual home healthcare. A total of 117 patients with T18 admitted to nine institutions between 2000 and 2015 were retrospectively investigated. After excluding four patients whose outcomes were unclear, we divided 113 patients into two groups-the survival discharge group (n = 52) and the death discharge group (n = 61)-and compared maternal factors, perinatal factors, neonatal factors, and therapeutic factors between the groups. In addition, home healthcare, readmission, utilization of respite care and home nursing, and cause of death among the survival group were surveyed. Fifty-two (44%) patients with T18 survived at discharge and their 1-year survival rate was 29%. The survival group had a longer gestation period, larger physique, and longer survival time, compared to the death group. Independent factors associated with survival discharge were the absence of an extremely low birthweight infant (ELBWI), the absence of esophageal atresia and patent ductus arteriosus, and cardiovascular surgery. All surviving patients required some home healthcare. The most frequent cause of death was a respiratory disorder. We recommend discussing the treatment strategy with families in the presence of neonatologists or pediatric surgeons, who can explain differences in prognosis, based on the gestation period, birthweight, severity of cardiovascular disease, and cardiovascular surgery.

Gender differences in adolescents' perceptions toward dentists using the Japanese version of the dental beliefs survey: a cross-sectional survey.

BACKGROUND: While adult women show greater dental anxiety than adult men, few studies have examined gender differences in adolescent perceptions of dentists. Therefore, this cross-sectional study aimed to evaluate the gender differences in adolescents' perceptions toward dentists by using the Japanese version of the Dental Beliefs Survey (DBS) and the factor structure of the DBS. METHODS: We conducted surveys at schools, and 957 Japanese adolescents (403 girls and 554 boys, aged between 13 and 15 years) participated in this study. To assess their confidence in dentists, participants were asked to complete the self-reported, 15-item Japanese version of the DBS. We performed a Welch's t-test and a one-way analysis of variance to assess differences in DBS scores by gender and age. Factor analysis (principal components, varimax rotation) was used to assess the scale's factor structure. RESULTS: A significant gender difference was observed in the DBS scores (P = 0.018), suggesting that boys exhibit greater negative perceptions toward the behavior of dentists than girls. However, there was no significant difference found among ages. The factor analysis yielded two results: Factor 1, "trust" (seven items); and Factor 2, "lack of control" (five items). Notably, the factor structure differed according to gender. As such, by including only factors with eigenvalues above 1.0, the DBS for girls comprised "trust" (seven items) and "communication" (three items), while that for boys comprised "lack of control" (six items) and "belittlement" (six items). CONCLUSIONS: This study identified two factors of differing strengths pertaining to the confidence of Japanese adolescents in dentists. Gender differences in perceptions toward dentists were observed. Accounting for these differences may improve the effectiveness of strategies to lower dental anxiety and foster positive dental beliefs in young patients.

[Immune-mediated neuropathy with anti-ganglioside antibodies in diffuse large B-cell lymphoma].

A 70-year-old man having a mass lesion on his right lower abdomen for 2 months was admitted to our hospital for diagnosis. Upon admission, the patient experienced bilateral upper and lower limb weakness, which aggravated. He underwent nerve conduction study and was diagnosed with axonal neuropathy. Diagnosis of diffuse large B-cell lymphoma (DLBCL) was accomplished via biopsy of the mass lesion, with positive laboratory tests for anti-ganglioside antibodies. Based on these results, immune-mediated DLBCL-induced polyneuropathy was suspected, and chemotherapy (R-CHOP) was immediately started. Limb weakness improved and completely resolved. After six courses of R-CHOP, no evidence of DLBCL was observed on PET/CT (i.e., complete metabolic remission). The patient lived without DLBCL relapse or neurological symptoms after remission. Only few reports regarding immune-mediated polyneuropathy induced by malignant lymphoma are available in the literature, which, together with this case, suggest that prompt control of malignant lymphoma is crucial for favorable prognosis of neuropathy.

Pretreatment evaluation of fluorescence resonance energy transfer-based drug sensitivity test for patients with chronic myelogenous leukemia treated with dasatinib.

Tyrosine kinase inhibitors (TKI) are used for primary therapy in patients with newly diagnosed CML. However, a reliable method for optimal selection of a TKI from the viewpoint of drug sensitivity of CML cells has not been established. We have developed a FRET-based drug sensitivity test in which a CrkL-derived fluorescent biosensor efficiently quantifies the kinase activity of BCR-ABL of living cells and sensitively evaluates the inhibitory activity of a TKI against BCR-ABL. Here, we validated the utility of the FRET-based drug sensitivity test carried out at diagnosis for predicting the molecular efficacy. Sixty-two patients with newly diagnosed chronic phase CML were enrolled in this study and treated with dasatinib. Bone marrow cells at diagnosis were subjected to FRET analysis. The ΔFRET value was calculated by subtraction of FRET efficiency in the presence of dasatinib from that in the absence of dasatinib. Treatment response was evaluated every 3 months by the BCR-ABL1 International Scale. Based on the ΔFRET value and molecular response, a threshold of the ΔFRET value in the top 10% of FRET efficiency was set to 0.31. Patients with ΔFRET value ≥0.31 had significantly superior molecular responses (MMR at 6 and 9 months and both MR4 and MR4.5 at 6, 9, and 12 months) compared with the responses in patients with ΔFRET value <0.31. These results suggest that the FRET-based drug sensitivity test at diagnosis can predict early and deep molecular responses. This study is registered with UMIN Clinical Trials Registry (UMIN000006358).

Temperature enhanced succinate production concurrent with increased central metabolism turnover in the cyanobacterium Synechocystis sp. PCC 6803.

Succinate is a versatile petrochemical compound that can be produced by microorganisms, often from carbohydrate based carbon sources. Phototrophic cyanobacteria including Synechocystis sp. PCC 6803 can more efficiently produce organic acids such as succinate without sugar supplementation, via photosynthetic production of glycogen followed by glycogen utilization, typically under dark conditions. In this study, Synechocystis 6803 bioproduction of organic acids under dark anoxic conditions was found to increase with elevation of temperature from 30 °C to 37 °C. The further enhancement of succinate bioproduction by overexpression of the rate limiting enzyme phosphoenolpyruvate carboxylase resulted in improved glycogen utilization. To gain more insight into the mechanisms underlying the increased organic acid output, a novel temperature dependent metabolomics analysis was performed. Adenylate energy charge was found to decrease along with elevating temperature, while central metabolites glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, glycerol 3-phosphate, malate, fumarate and succinate increased. Temperature dependent 13C-labeling metabolomics analysis further revealed a glycolysis to TCA bottleneck, which could be overcome by addition of CO2, leading to even higher organic acid production. Optimization of initial cell concentration to 25 g-dry cell weight/L, in combination with 100 mM NaHCO3 supplementation, afforded a succinate titer of over 1.8 g/L, the highest reported autotrophic succinate titer. Succinate titers remained high after additional knockout of ackA, resulting in the highest reported autotrophic D-lactate titer as well. The optimization of Synechocystis 6803 organic acid production therefore holds significant promise for CO2 capture and utilization.

Molecular Characterization of Prototheca strains isolated in China revealed the first cases of protothecosis associated with Prototheca zopfii genotype 1.

In this study, six strains of Prototheca isolated in China from human patients diagnosed as protothecosis and cows with mastitis were characterized by polymerase chain reaction (PCR) and nucleotide sequencing of the ribosomal RNA gene (rDNA) and by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The results indicated that three strains isolated from the human patients were P. zopfii genotype 1, revealing the first cases of human protothecosis associated with P. zopfii genotype 1. The remaining three strains were shown to be P. zopfii genotype 2. Interestingly, one strain isolated from the cerebrospinal fluid of the human patient appeared to have both of the genotype 1- and 2-specific alleles in the small subunit (SSU) rDNA although it was classified by MALDI-TOF MS as genotype 2. For genotyping of certain strains of P. zopfii, it may be necessary to comprehensively evaluate the diversity in the SSU rDNA sequences and the MALDI-TOF MS results.