Absence of KpsM (Slr0977) Impairs the Secretion of Extracellular Polymeric Substances (EPS) and Impacts Carbon Fluxes in Synechocystis sp. PCC 6803

ABSTRACT Many cyanobacteria produce extracellular polymeric substances (EPS), composed mainly of heteropolysaccharides, that play a variety of physiological roles, being crucial for cell protection, motility, and biofilm formation. However, due to their complexity, the EPS biosynthetic pathways as well as their assembly and export mechanisms are still far from being fully understood. Here, we show that the absence of a putative EPS-related protein, KpsM (Slr0977), has a pleiotropic effect on Synechocystis sp. strain PCC 6803 physiology, with a strong impact on the export of EPS and carbon fluxes. The kpsM mutant exhibits a significant reduction of released polysaccharides and a smaller decrease of capsular polysaccharides, but it accumulates more polyhydroxybutyrate (PHB) than the wild type. In addition, this strain shows a light/cell density-dependent clumping phenotype and exhibits an altered protein secretion capacity. Furthermore, the most important structural component of pili, the protein PilA, was found to have a modified glycosylation pattern in the mutant compared to the wild type. Proteomic and transcriptomic analyses revealed significant changes in the mechanisms of energy production and conversion, namely, photosynthesis, oxidative phosphorylation, and carbon metabolism, in response to the inactivation of slr0977. Overall, this work shows for the first time that cells with impaired EPS secretion undergo transcriptomic and proteomic adjustments, highlighting the importance of EPS as a major carbon sink in cyanobacteria. The accumulation of PHB in cells of the mutant, without affecting significantly its fitness/growth rate, points to its possible use as a chassis for the production of compounds of interest. IMPORTANCE Most cyanobacteria produce extracellular polymeric substances (EPS) that fulfill different biological roles depending on the strain/environmental conditions. The interest in the cyanobacterial EPS synthesis/export pathways has been increasing, not only to optimize EPS production but also to efficiently redirect carbon flux toward the production of other compounds, allowing the implementation of industrial systems based on cyanobacterial cell factories. Here, we show that a Synechocystis kpsM (slr0977) mutant secretes less EPS than the wild type, accumulating more carbon intracellularly, as polyhydroxybutyrate. Further characterization showed a light/cell density-dependent clumping phenotype, altered protein secretion, and modified glycosylation of PilA. The proteome and transcriptome of the mutant revealed significant changes, namely, in photosynthesis and carbon metabolism. Altogether, this work provides a comprehensive overview of the impact of kpsM disruption on Synechocystis physiology, highlighting the importance of EPS as a carbon sink and showing how cells adapt when their secretion is impaired, and the redirection of the carbon fluxes.

Red fluorescent carbon dots excited by visible light: cell imaging and visual detection of ammonia gas using PVB films

Red fluorescent carbon dots are used in cell imaging and detection of ammonia gas using PVB films under visible light.

PATHOMORPHOLOGICAL ANALYSIS OF THE LIGHT-CELL VARIANT OF RENAL CELL CARCINOMA

Renal cell carcinoma (kidney cancer, RCC) has been and remains one of the most important problems of oncourology, which is expressed by a steady increase in the incidence of 6—10 % every year [1]. Pathology occurs with the same frequency in both men (2.4 % in the structure of cancer mortality in the Republic of Belarus) and women (2.1 %) and accounts for 4 % of all adult malignant tumors. Objective. To identify the diagnostically significant pathomorphological aspects of the light-cell variant of renal cell carcinoma by morphometric analysis. Materials and methods. We analyzed the surgical material from 20 patients (10 men and 10 women) aged 29 to 83 years (median 64 ± 3.2 years) with a verified diagnosis of renal cell carcinoma from the State Institution "RNPC OMR named after N. N. Alexandrov". Grading and staging of the tumor was carried out according to the recommendations of the WHO (World Health Organization Classification of Tumours. Pathology & Genetics: Tumours of the Urinary System and Male Genital Organs. Lyon, France: IARC Press, 2004:179—184). The pathomorphological assessment of the material was carried out both at the microscopic and macro levels. Methods used: histological and morphometric. Results. At the macro level, in all cases of SPCR, the growth patterns, cytological and nuclear signs characteristic of it were revealed: the average size of the cells: 5,0*6,7*4,8 cm (p < 0.05), the material is light yellow with areas of hemorrhage. Macroscopically, it can be seen that in most cases the tumor extends into the renal sinus and a smooth contour into the paranephral tissue. CHLS invasion was detected in one case (5 %), invasion into the portal vessels — in two cases (10 %), focal compression of CHLS — in one case (5 %). The parenchyma is not preserved along the entire perimeter in all cases of SPCR, and PPCR (p > 0.1). Conclusion: 1. According to the results of the work carried out, it was found that the light-cell variant of renal cell carcinoma is characterized by a variety of pathomorphological manifestations that occur in other variants of renal cancer. On the other hand, the presence of foci of "thyroidization" was detected only in SPCR. 2. The morphometric parameters of the nuclei of renal cell carcinoma cells may directly depend on the patient's gender. In this case, the superiority of nuclei in size was observed in male patients. 3. The nuclei in the light-cell variant of renal cancer are significantly larger than the nuclei in chromophobic renal cancer and smaller than the cell nuclei in papillary renal cancer. 4. These indicators can be used in the future to improve the definition of significant criteria for the differential diagnosis of various variants of renal cell cancers.

Active monitoring of contrast-accumulating kidney tumours

Introduction. The incidence of kidney cancer (KC) in the world is increasing and today is about 3%, but the death rate from this type of malignancy does not increase proportionally. According to research by many authors, more than half of the patients are over 65 years old at the time of diagnosis. Patients at this age have a high incidence of high comorbidity and risk of death from cardiovascular or other intercurrent pathology that exceeds the risk of death from KC. Recently, there has been a positive trend in the detection of the disease in the early stages up to 61.80%. Most of the primary detected kidney tumours are diagnosed randomly as asymptomatically small (less than 4 cm) tumours without signs of visceral metastasis. These tumours have a high degree of differentiation and rarely require surgical treatment in addition to their small size, and in the case of surgery, these pathomorphological results are benign. Due to the slow progredient growth of kidney formations and an asymptomatic course, the method of dynamic observation of kidney tumours is relevant in elderly patients and avoids unnecessary risks of surgical treatment of localized KC.Purpose of the study. To trace the growth rate of kidney tumours accumulating contrast agent using the method of dynamic observation. This study will allow us to differentially approach the choice of surgical treatment, which is optimal for elderly patients with low somatic status.Materials and methods. In the Multidisciplinary clinical medical centre «Medical City» (Tyumen) database all cases of radiographically verified space-occupying lesions of the kidneys that accumulate contrast were selected in the period from 2009 to 2019. We studied 50 people: 23 women (46%) and 27 men (54%), aged from 58 to 90 years. The study group included patients with kidney neoplasms of size < 7cm. Patients whose follow-up period was less than 12 months were excluded from the analysis. Regularly, every 3 to 6 months, patients underwent computed tomography to assess the growth dynamics. The size of the tumour, which was assumed to be its diameter in the largest dimension, was carefully studied. The growth rate of the tumour was calculated as the average change in diameter for 1 year during the entire observation period.Results. The average age of patients was 74.8 ± 7.4 years according to the results of the study. The age of patients at the time of diagnosis also had no prognostic significance for the rate of growth of kidney tumours (p > 0.05). The primary diagnosis was made in 32 patients (64%) using CT, in 18 (36%) using ultrasound. The average size of the tumou at the time of detection was 35.0 ± 6.9 mm. Percutaneous kidney biopsy was performed in 2 patients for morphological verification of the tumour type. Moderate-differentiated light-cell renal cell carcinoma pT1bN0M0 was detected in both patients according to the results of histological differentiation. The average linear growth rate of the tumour was 6.6 ± 2.4 mm / year. The size of the tumour at the time of diagnosis was not correlated with the growth rate (p > 0.05). There was no correlation between the rate of increase in the size of formations depending on their structure — solid (median 6 mm / year; average — 10 mm / year) or cystic-solid (median 7 mm / year; average 9 mm / year; p > 0.05). The absence of tumour growth dynamics during the entire observation period was detected in 22 (44%) people, including 10 (20%) men and 12 (24%) women. Visceral metastasis was diagnosed in 3 cases: to the liver, spleen, and the appearance of a second tumour on the contralateral kidney. Surgical treatment was performed in 4 patients (8%), in 2 (4%) cases, the indication for surgery was the progression of the tumour in the form of the appearance of visceral metastases. One patient had chromophobic KC pT1bN0M1, the other had renal cell carcinoma, a light-cell variant of pT1bN0M1. The operation in the volume of kidney resection was performed in 2 (4%) patients, in both cases, morphologically confirmed renal cell carcinoma, light-cell variant pT1aN0M0. The presence of a cystic-solid component and the initial size of the tumour were potential radiographic signs that could predict the dynamics of an increase in renal parenchyma neoplasm.Conclusion. The tactic of actively observing the growth rates of kidney tumours that accumulate contrast material allowed us to better understand the biological behaviour of KC. It was found that most kidney malignancies have a slow growth rate when determining the linear growth rate of the tumour. This conclusion allows us to differentially approach the choice of surgical treatment, which is optimal for elderly patients with low somatic status. Because prognostic signs of KC have not yet been identified and are not fixed in international treatment protocols, all patients who are suitable candidates for surgery are shown operative treatment.