Collagen Lattice Model, Populated with Heterogeneous Cancer-Associated Fibroblasts, Facilitates Advanced Reconstruction of Pancreatic Cancer Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is a solid-tumor malignancy. To enhance the treatment landscape of PDAC, a 3D model optimized for rigorous drug screening is essential. Within the PDAC tumor microenvironment, a dense stroma comprising a large extracellular matrix and cancer-associated fibroblasts (CAFs) is well-known for its vital role in modulating tumor growth, cellular heterogeneity, bidirectional paracrine signaling, and chemoresistance. In this study, we employed a fibroblast-populated collagen lattice (FPCL) modeling approach that has the ability to replicate fibroblast contractility in the collagenous matrix to build dense stroma. This FPCL model allows CAF differentiation by facilitating multifaceted cell-cell interactions between cancer cells and CAFs, with the differentiation further influenced by mechanical forces and hypoxia carried within the 3D structure. Our FPCL models displayed hallmark features, including ductal gland structures and differentiated CAFs with spindle shapes. Through morphological explorations alongside in-depth transcriptomic and metabolomic profiling, we identified substantial molecular shifts from the nascent to mature model stages and potential metabolic biomarkers, such as proline. The initial pharmacological assays highlighted the effectiveness of our FPCL model in screening for improved therapeutic strategies. In conclusion, our PDAC modeling platform mirrors complex tumor microenvironmental dynamics and offers an unparalleled perspective for therapeutic exploration.

Establishment of a zebrafish inbred strain, M-AB, capable of regular breeding and genetic manipulation.

Inbred strains of organisms are genetically highly uniform and thus useful for life science research. We have previously reported the ongoing generation of the zebrafish IM strain from the India (IND) strain through full sib-pair mating for 16 generations. However, the IM fish laid a small number of offspring and had a short lifespan, implying the need for discreet care in breeding. Here, we report the subsequent establishment of IM strain as well as the generation of a new inbred zebrafish strain, Mishima-AB (M-AB). M-AB was derived from the *AB strain by full sib-pair mating for over 20 generations, which fulfills the general criterion for the establishment of an inbred strain. In contrast to the IM case, maintenance of the M-AB strain by sib-pair mating required almost no special handling. Genome sequencing of IM individuals from the 47th generation and M-AB individuals from the 27th generation revealed that SNP-based genomic heterogeneity across whole-genome nucleotides was 0.008% and 0.011%, respectively. These percentages were much lower than those of the parental IND (0.197%) and *AB (0.086%) strains. These results indicate that the genomes of these inbred strains were highly homogenous. We also demonstrated the successful microinjection of antisense morpholinos, CRISPR/Cas9, and foreign genes into M-AB embryos at the 1-cell stage. Overall, we report the establishment of a zebrafish inbred strain, M-AB, which is capable of regular breeding and genetic manipulation. This strain will be useful for the analysis of genetically susceptible phenotypes such as behaviors, microbiome features and drug susceptibility.

Increased skeletal muscle mass index was involved in glycemic efficacy following diabetes treatment, and changes in fat mass index correlated with the changes in the lipid ratio in type 2 diabetes.

AIM: This study aimed to investigate the association between changes in body composition, glycated hemoglobin, and lipid ratio during the treatment of patients with type 2 diabetes mellitus (T2DM). METHODS: This retrospective analysis used data from outpatients with T2DM who had confirmed body composition and measured laboratories at administration and after treatment. The baseline characteristics and prescribed treatment were collected. The total cholesterol/high-density lipoprotein cholesterol (HDL) ratio, low-density lipoprotein cholesterol (LDL)/HDL ratio, and triglyceride-glucose (TyG) index were also calculated. RESULTS: A total of 207 patients (mean patient age, 62.0 ± 13.7 years; 68.1 % males) were enrolled. Fat mass index (FMI) changes correlated with the changes in the lipid ratio, whereas skeletal muscle mass index (SMI) changes inversely correlated with glycated hemoglobin (HbA1c) changes. Multiple regression analysis showed that changes in LDL/HDL and TyG correlated with FMI changes (t = 2.388, p = 0.017, t = 2.022, p = 0.044). Conversely, HbA1c changes correlated with SMI changes (t = -2.552, p = 0.011). CONCLUSION: In patients with T2DM, increased SMI was involved in glycemic efficacy, and FMI changes were associated with LDL/HDL and TyG.

Therapeutic Potential of Adipose-Derived Stem Cell-Conditioned Medium and Extracellular Vesicles in an In Vitro Radiation-Induced Skin Injury Model.

Radiotherapy (RT) is one of three major treatments for malignant tumors, and one of its most common side effects is skin and soft tissue injury. However, the treatment of these remains challenging. Several studies have shown that mesenchymal stem cell (MSC) treatment enhances skin wound healing. In this study, we extracted human dermal fibroblasts (HDFs) and adipose-derived stem cells (ADSCs) from patients and generated an in vitro radiation-induced skin injury model with HDFs to verify the effect of conditioned medium derived from adipose-derived stem cells (ADSC-CM) and extracellular vesicles derived from adipose-derived stem cells (ADSC-EVs) on the healing of radiation-induced skin injury. The results showed that collagen synthesis was significantly increased in wounds treated with ADSC-CM or ADSC-EVs compared with the control group, which promoted the expression of collagen-related genes and suppressed the expression of inflammation-related genes. These findings indicated that treatment with ADSC-CM or ADSC-EVs suppressed inflammation and promoted extracellular matrix deposition; treatment with ADSC-EVs also promoted fibroblast proliferation. In conclusion, these results demonstrate the effectiveness of ADSC-CM and ADSC-EVs in the healing of radiation-induced skin injury.

In Vitro Storage of Functional Sperm at Room Temperature in Zebrafish and Medaka.

The longevity of sperm in teleost such as zebrafish and medaka is short when isolated even in saline-balanced solution at a physiological temperature. In contrast, some internal fertilizers exhibit the long-term storage of sperm, >10 months, in the female reproductive tract. This evidence implies that sperm in teleost possesses the ability to survive for a long time under suitable conditions; however, these conditions are not well understood. In this study, we show that the sperm of zebrafish can survive and maintain fertility in L-15-based storage medium supplemented with bovine serum albumin, fetal bovine serum, glucose, and lactic acid for 28 days at room temperature. The fertilized embryos developed to normal fertile adults. This storage medium was effective in medaka sperm stored for 7 days at room temperature. These results suggest that sperm from external fertilizer zebrafish and medaka has the ability to survive for at least 4 and 1 week, respectively, in the body fluid-like medium at a physiological temperature. This sperm storage method allows researchers to ship sperm by low-cost methods and to investigate key factors for motility and fertile ability in those sperm.

Fatty liver index predicts the development of hypertension in a Japanese general population with and without dysglycemia.

Fatty liver has been suggested to be associated with the development of hypertension. However, whether this association is related to glycemia has not been elucidated. Therefore, we investigated whether the fatty liver index (FLI) predicts the development of hypertension among individuals with and without dysglycemia in a general Japanese population. A total of 3114 participants (1036 males and 2078 females) without hypertension who underwent a Specific Health Checkup in the fiscal year 2013 were followed up until 2018. The participants were divided into six groups based on FLI tertiles (low, moderate, or high) and whether they had dysglycemia. We estimated the hazard ratios (HRs) of each group by sex using the Cox proportional hazard model. Models were adjusted for age, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, smoking, and alcohol consumption and further adjusted for systolic blood pressure (SBP). During the mean follow-up period of 2.8 years, 160 of the 3114 participants developed hypertension. Using the low FLI group with normoglycemia as a reference, the HR for incident hypertension was increased in the high FLI group with and without dysglycemia in both sexes after adjusting for confounders, except SBP (HR [95% confidence interval]: male: 1.52 (1.06-2.17) in normoglycemia and 2.05 (1.43-2.92) in dysglycemia, and female: 1.86 (1.43-2.42) in normoglycemia and 2.98 (2.19-4.07) in dysglycemia). Furthermore, in females, this association was observed after adjusting for SBP. We concluded that FLI was independently associated with an increased risk of incident hypertension in individuals with and without dysglycemia.

Impact of hypertension and diabetes on the onset of chronic kidney disease in a general Japanese population.

Hypertension (HT) and diabetes mellitus (DM) are both major risk factors for chronic kidney disease (CKD); however, few studies have examined the impacts of the combination of HT and DM on CKD development in general populations. We aimed to explore whether HT or DM contributes more to CKD development in a Japanese community. A total of 5823 individuals without a history of CKD who underwent specific health checkups in fiscal year 2013 were monitored until the end of March 2018. Participants were categorized as having neither HT nor DM (none group), either HT or DM, and both (HT + DM). We calculated the hazard ratios (HRs) for developing CKD in each category using Cox proportional hazards models after adjusting for age, dyslipidemia, smoking, and alcohol drinking and with the none group as the reference. We also estimated the population attributable fraction (PAF) for CKD development in populations with either HT or DM or both. During a mean follow-up of 3.0 years, 759 individuals developed CKD, with HRs of 1.56 with a 95% confidence interval (CI) [1.33, 1.83], 1.22 with a 95% CI [0.86, 1.75], and 2.83 with a 95% CI [2.22, 3.63] for the HT only, DM only and HT + DM categories, respectively. Sex-specific analysis showed similar findings. The PAFs for CKD (14.1% and 17.2% for men and women, respectively) were the highest among participants with HT only. We concluded that in this Japanese community, HT contributed more than DM to CKD development; hence, managing hypertension is important to prevent CKD as well as diabetes.

Therapeutic Potential of Adipose Stem Cell-Derived Conditioned Medium on Scar Contraction Model.

Scars are composed of stiff collagen fibers, which contract strongly owing to the action of myofibroblasts. To explore the substances that modulate scar contracture, the fibroblast-populated collagen lattice (FPCL) model has been used. However, the molecular signature of the patient-derived FPCL model has not been verified. Here, we examined whether the patient-derived keloid FPCL model reflects scar contraction, analyzing detailed gene expression changes using comprehensive RNA sequencing and histological morphology, and revealed that these models are consistent with the changes during human scar contracture. Moreover, we examined whether conditioned media derived from adipose stem cells (ASC-CM) suppress the scar contracture of the collagen disc. Detailed time-series measurements of changes in disc area showed that the addition of ASC-CM significantly inhibited the shrinkage of collagen discs. In addition, a deep sequencing data analysis revealed that ASC-CM suppressed inflammation-related gene expression in the early phase of contraction; in the later phase, this suppression was gradually replaced by extracellular matrix (ECM)-related gene expression. These lines of data suggested the effectiveness of ASC-CM in suppressing scar contractures. Therefore, the molecular analysis of the ASC-CM actions found in this study will contribute to solving medical problems regarding pathological scarring in wound prognosis.

Sarcopenia is associated with the Geriatric Nutritional Risk Index in elderly patients with poorly controlled type 2 diabetes mellitus.

AIMS/INTRODUCTION: Diabetes and sarcopenia have a two-way relationship with each other with advanced age. Additionally, malnutrition is correlated with a higher risk of sarcopenia in elderly patients. This study evaluated the association between sarcopenia and geriatric nutritional risk index (GNRI) in elderly patients with type 2 diabetes mellitus. MATERIALS AND METHODS: Patients with type 2 diabetes mellitus aged ≥60 years were recruited from June 2018 to August 2020. This study analyzed 234 patients, who completed a physical performance test required for the diagnosis of sarcopenia. To investigate the effect of GNRI on sarcopenia, logistic regression analyses was used. RESULTS: Patients with sarcopenia were significantly older with a lower body mass index (BMI) and GNRI compared with normal patients. The GNRI showed a positive correlation with the skeletal muscle index (SMI) and handgrip strength (SMI: R = 0.486, P < 0.001 for male; R = 0.589, P < 0.001 for female, handgrip strength: R = 0.470, P < 0.001 for male, R = 0.364, P < 0.001 for female). In the multivariate logistic regression model, a higher GNRI was associated with a lower risk of sarcopenia in older men and women with diabetes (adjusted odds ratio [OR], 0.892; 95% confidence interval [CI], 0.839-0.948 for male; adjusted OR, 0.928; 95% CI, 0.876-0.982 for female). One year of diabetes treatment improved the GNRI in the sarcopenia group with type 2 diabetes mellitus. CONCLUSIONS: A low GNRI was associated with an increased risk of sarcopenia in elderly patients with type 2 diabetes mellitus. Treatment with glucose-lowering drugs improved the GNRI in the sarcopenia group.

Prediction of Lifetime Risk of Cardiovascular Disease Deaths Stratified by Sex in the Japanese Population.

Background Lifetime risk is an informative estimate for driving lifestyle and behavioral changes especially for young adults. The impact of composite risk factors for cardiovascular disease on lifetime risk stratified by sex has not been investigated in the Japanese population, which has a much lower mortality of coronary heart disease compared with the Western population. We aimed to estimate lifetime risk of death from cardiovascular disease attributable to traditional risk factors. Methods and Results We analyzed pooled individual data from the Evidence for Cardiovascular Prevention from Observational Cohorts in a Japanese cohort study. A modified Kaplan-Meier approach was used to estimate the remaining lifetime risk of cardiovascular death. In total, 41 002 Japanese men and women with 537 126 person-years of follow-up were included. The lifetime risk at the index-age of 45 years for those with optimal risk factors (total cholesterol <4.65 mmol/L, systolic blood pressure <120 mm Hg, diastolic blood pressure <80 mm Hg, absence of diabetes, and absence of smoking habit) was lower compared with the highest risk profile of ≥2 risk factors (6.8% [95% CI, 0%-11.9%] versus 19.4% [16.7%-21.4%] for men and 6.9% [1.2%-11.5%] versus 15.4% [12.6%-18.1%] for women). Conclusions The magnitude and the number of risk factors were progressively associated with increased lifetime risk even in individuals in early adulthood who tend to have low short-term risk. The degree of established cardiovascular risk factors can be converted into lifetime risk. Our findings may be useful for risk communication in the early detection of future cardiovascular disease risk.

Meiotic Chromosome Dynamics in Zebrafish.

Recent studies in zebrafish have revealed key features of meiotic chromosome dynamics, including clustering of telomeres in the bouquet configuration, biogenesis of chromosome axis structures, and the assembly and disassembly of the synaptonemal complex that aligns homologs end-to-end. The telomere bouquet stage is especially pronounced in zebrafish meiosis and sub-telomeric regions play key roles in mediating pairing and homologous recombination. In this review, we discuss the temporal progression of these events in meiosis prophase I and highlight the roles of proteins associated with meiotic chromosome architecture in homologous recombination. Finally, we discuss the interplay between meiotic mutants and gonadal sex differentiation and future research directions to study meiosis in living cells, including cell culture.

Sycp1 Is Not Required for Subtelomeric DNA Double-Strand Breaks but Is Required for Homologous Alignment in Zebrafish Spermatocytes.

In meiotic prophase I, homologous chromosomes are bound together by the synaptonemal complex, in which two axial elements are connected by transverse filaments and central element proteins. In human and zebrafish spermatocytes, homologous recombination and assembly of the synaptonemal complex initiate predominantly near telomeres. In mice, synapsis is not required for meiotic double-strand breaks (DSBs) and homolog alignment but is required for DSB repair; however, the interplay of these meiotic events in the context of peritelomeric bias remains unclear. In this study, we identified a premature stop mutation in the zebrafish gene encoding the transverse filament protein Sycp1. In sycp1 mutant zebrafish spermatocytes, axial elements were formed and paired at chromosome ends between homologs during early to mid-zygonema. However, they did not synapse, and their associations were mostly lost in late zygotene- or pachytene-like stages. In sycp1 mutant spermatocytes, γH2AX signals were observed, and Dmc1/Rad51 and RPA signals appeared predominantly near telomeres, resembling wild-type phenotypes. We observed persistent localization of Hormad1 along the axis in sycp1 mutant spermatocytes, while the majority of Iho1 signals appeared and disappeared with kinetics similar to those in wild-type spermatocytes. Notably, persistent Iho1 foci were observed in spo11 mutant spermatocytes, suggesting that Iho1 dissociation from axes occurs in a DSB-dependent manner. Our results demonstrated that Sycp1 is not required for peritelomeric DSB formation but is necessary for complete pairing of homologs in zebrafish meiosis.

PRDM9 activity depends on HELLS and promotes local 5-hydroxymethylcytosine enrichment.

Meiotic recombination starts with the formation of DNA double-strand breaks (DSBs) at specific genomic locations that correspond to PRDM9-binding sites. The molecular steps occurring from PRDM9 binding to DSB formation are unknown. Using proteomic approaches to find PRDM9 partners, we identified HELLS, a member of the SNF2-like family of chromatin remodelers. Upon functional analyses during mouse male meiosis, we demonstrated that HELLS is required for PRDM9 binding and DSB activity at PRDM9 sites. However, HELLS is not required for DSB activity at PRDM9-independent sites. HELLS is also essential for 5-hydroxymethylcytosine (5hmC) enrichment at PRDM9 sites. Analyses of 5hmC in mice deficient for SPO11, which catalyzes DSB formation, and in PRDM9 methyltransferase deficient mice reveal that 5hmC is triggered at DSB-prone sites upon PRDM9 binding and histone modification, but independent of DSB activity. These findings highlight the complex regulation of the chromatin and epigenetic environments at PRDM9-specified hotspots.

Impact of hypertension stratified by diabetes on the lifetime risk of cardiovascular disease mortality in Japan: a pooled analysis of data from the Evidence for Cardiovascular Prevention from Observational Cohorts in Japan study.

Lifetime risk is an informative estimate to motivate people to change lifestyle behaviors, especially from a younger age, in public health education. The impact of the combination of hypertension and diabetes on the lifetime risk of cardiovascular mortality has not been investigated in Asian populations. A pooled analysis of individual data from nine cohorts was performed. A total of 57,339 Japanese men and women were eligible for the analysis. We used the modified Kaplan-Meier approach and estimated the remaining lifetime risk of cardiovascular mortality starting from the index age of 35 years. Participants were classified into four categories defined by hypertension and diabetes. The lifetime risk was increased in the order of those without either risk, those without hypertension but with diabetes, those with hypertension but without diabetes, and those with both risks. The lifetime risk of cardiovascular mortality at the 35-year index age was as follows: 7.8% in men and 6.2% in women for those without either hypertension or diabetes, 13.2% in men and 9.5% in women for those without hypertension but with diabetes, 17.2% in men and 11.7% in women for those with hypertension but without diabetes, and 19.4% in men and 15% in women for those with both risks. These findings reinforce the need for a life-course perspective in the management of hypertension and diabetes from a younger age.

Fabrication of Perfusable Vascular Channels and Capillaries in 3D Liver-like Tissue.

Although various production methods for 3D vascularised tissues have been developed, constructing capillary-like structures branching from perfusable large channels remains difficult. This study describes a method to fabricate tube-shaped 3D liver-like tissue (tubular liver tissue) with large channels and capillary-like structures using a perfusion device. The perfusion device functions as an interface between the tissue and an external pump, as it has connectors equipped with anchors that hold the tissue in response to its shrinkage, which is accompanied by the self-organisation of capillary-like structures. Histological analysis revealed that perfusion via the large channel induced capillary formation around the channel and maintained proper tissue functions. Accompanied by structural examinations, global gene expression analysis supported this finding; specifically, genes involved in angiogenesis were enriched in the perfused condition. Furthermore, we confirmed the penetrability of the capillary-like structures by infusing India ink, as well as substance exchange by measuring the amounts of secreted albumin. These lines of evidence indicate that our method can be used to construct 3D tissues, which is useful for fields of in vitro tissue regeneration for drug development and regenerative medicine.

Sycp2 is essential for synaptonemal complex assembly, early meiotic recombination and homologous pairing in zebrafish spermatocytes.

Meiotic recombination is essential for faithful segregation of homologous chromosomes during gametogenesis. The progression of recombination is associated with dynamic changes in meiotic chromatin structures. However, whether Sycp2, a key structural component of meiotic chromatin, is required for the initiation of meiotic recombination is still unclear in vertebrates. Here, we describe that Sycp2 is required for assembly of the synaptonemal complex and early meiotic events in zebrafish spermatocytes. Our genetic screening by N-ethyl-N-nitrosourea mutagenesis revealed that ietsugu (its), a mutant zebrafish line with an aberrant splice site in the sycp2 gene, showed a defect during meiotic prophase I. The its mutation appeared to be a hypomorphic mutation compared to sycp2 knockout mutations generated by TALEN mutagenesis. Taking advantage of these sycp2 hypomorphic and knockout mutant lines, we demonstrated that Sycp2 is required for the assembly of the synaptonemal complex that is initiated in the vicinity of telomeres in wild-type zebrafish spermatocytes. Accordingly, homologous pairing, the foci of the meiotic recombinases Dmc1/Rad51 and RPA, and γH2AX signals were largely diminished in sycp2 knockout spermatocytes. Taken together, our data indicate that Sycp2 plays a critical role in not only the assembly of the synaptonemal complex, but also early meiotic recombination and homologous pairing, in vertebrates.

The PRDM9 KRAB domain is required for meiosis and involved in protein interactions.

PR domain-containing protein 9 (PRDM9) is a major regulator of the localization of meiotic recombination hotspots in the human and mouse genomes. This role involves its DNA-binding domain, which is composed of a tandem array of zinc fingers, and PRDM9-dependent trimethylation of histone H3 at lysine 4. PRDM9 is a member of the PRDM family of transcription regulators, but unlike other family members, it contains a Krüppel-associated box (KRAB)-related domain that is predicted to be a potential protein interaction domain. Here, we show that truncation of the KRAB domain of mouse PRDM9 leads to loss of PRDM9 function and altered meiotic prophase and gametogenesis. In addition, we identified proteins that interact with the KRAB domain of PRDM9 in yeast two-hybrid assay screens, particularly CXXC1, a member of the COMPASS complex. We also show that CXXC1 interacts with IHO1, an essential component of the meiotic double-strand break (DSB) machinery. As CXXC1 is orthologous to Saccharomyces cerevisiae Spp1 that links DSB sites to the DSB machinery on the chromosome axis, we propose that these molecular interactions involved in the regulation of meiotic DSB formation are conserved in mouse meiosis.

Effects of Daprodustat, a Novel Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor on Anemia Management in Japanese Hemodialysis Subjects.

BACKGROUND: Daprodustat (GSK1278863) is an oral hypoxia-inducible factor prolyl hydroxylase inhibitor being developed for treatment of anemia associated with chronic kidney disease (CKD). The effect of daprodustat in Japanese CKD patients with anemia has not been previously investigated. METHODS: We evaluated the relationship between daprodustat dose and hemoglobin response in Japanese patients on hemodialysis (HD) with anemia in a 4-week, phase II, double-blind, placebo-controlled study. After interrupting their erythropoiesis-stimulating agent for between 2 and 8 weeks, subjects with hemoglobin 8.5-10.5 g/dL were randomized to placebo or daprodustat 4, 6, 8, or 10 mg orally once daily. Hemoglobin, erythropoietin (EPO), and vascular endothelial growth factor (VEGF) levels during therapy were evaluated. RESULTS: Eighty-six of 97 randomized subjects completed the study. Mean baseline hemoglobin ranged from 9.68 to 9.92 g/dL across groups. After 4-week administration, mean hemoglobin changes were -0.28, -0.01, 0.54, and 0.97 g/dL in the 4, 6, 8, and 10 mg groups, respectively, as compared to -1.41 g/dL for placebo. Dose-dependent increase in plasma EPO concentration were observed up to 8 mg, with the 10 mg dose responses being similar to 8 mg. Plasma VEGF concentrations were minimally changed, even though 5 subjects treated with 6-10 mg reached EPO >500 mIU/mL. CONCLUSION: Daprodustat 4-10 mg once-daily produced dose-dependent increase in hemoglobin relative to placebo in Japanese HD subjects. The doses evaluated in the study have moderately increased endogenous EPO without changes in circulating VEGF levels.

Septal localization by membrane targeting sequences and a conserved sequence essential for activity at the COOH-terminus of Bacillus subtilis cardiolipin synthase.

The acidic phospholipid cardiolipin (CL) is localized on polar and septal membranes and plays an important physiological role in Bacillus subtilis cells. ClsA, the enzyme responsible for CL synthesis, is also localized on septal membranes. We found that GFP fusion proteins of the enzyme with NH2-terminal and internal deletions retained septal localization. However, derivatives with deletions starting from the COOH-terminus (Leu482) ceased to localize to the septum once the deletion passed the Ile residue at 448, indicating that the sequence responsible for septal localization is confined within a short distance from the COOH-terminus. Two sequences, Ile436-Leu450 and Leu466-Leu478, are predicted to individually form an amphipathic α-helix. This configuration is known as a membrane targeting sequence (MTS) and we therefore refer to them as MTS2 and MTS1, respectively. Either one has the ability to affect septal localization, and each of these sequences by itself localizes to the septum. Membrane association of the constructs of this enzyme containing the MTSs was verified by subcellular fractionation of the cells. CL synthesis, in contrast, was abolished after deleting just the last residue, Leu482, in the COOH-terminal four amino acid residue sequence, Ser-Pro-Ile-Leu, which is highly conserved among bacterial CL synthases.