T-cell immune profile in blood of systemic mastocytosis: Association with disease features.

BACKGROUND: Systemic mastocytosis (SM) is a heterogeneous disease characterized by an expansion of KIT-mutated mast cells (MC). KIT-mutated MC display activated features and release MC mediators that might act on the tumour microenvironment and other immune cells. Here, we investigated the distribution of lymphocyte subsets in blood of patients with distinct subtypes of SM and determined its association with other disease features. METHODS: We studied the distribution of TCD4+ and TCD4- cytotoxic cells and their subsets, as well as total NK- and B cells, in blood of 115 SM patients-38 bone marrow mastocytosis (BMM), 67 indolent SM (ISM), 10 aggressive SM (ASM)- and 83 age-matched healthy donors (HD), using spectral flow cytometry and the EuroFlow Immunomonitoring panel, and correlated it with multilineage KITD816V , the alpha-tryptasemia genotype (HαT) and the clinical manifestations of the disease. RESULTS: SM patients showed decreased counts (vs. HD) of TCD4- cytotoxic cells, NK cells and several functional subsets of TCD4+ cells (total Th1, Th2-effector memory, Th22-terminal effector and Th1-like Tregs), together with increased T-follicular-helper and Th1/Th17-like Treg counts, associated with different immune profiles per diagnostic subtype of SM, in multilineal versus MC-restricted KITD816V and in cases with a HαT+ versus HαT- genotype. Unique immune profiles were found among BMM and ISM patients with MC-restricted KITD816V who displayed HαT, anaphylaxis, hymenoptera venom allergy, bone disease, pruritus, flushing and GI symptoms. CONCLUSION: Our results reveal altered T- and NK-cell immune profiles in blood of SM, which vary per disease subtype, the pattern of involvement of haematopoiesis by KITD816V , the HαT genotype and specific clinical manifestations of the disease.

Obesity related pathophysiological aspects favouring Gastroesophageal Reflux Disease.

Gastroesophageal reflux disease (GERD) is a complex entity and one of the most frequents in general population. Association of GERD with obesity is clearly defined from an epidemiological point of view, where up to 40% of patients with overweight and obesity present reflux. Most of the risk factors associated to GERD are shared and especially frequent in patients with obesity. The pathophysiology that explain this correlation is complex and multifactorial, and includes both aspects related to physiology of motility, and anatomic changes. The malfunction of the lower esophageal sphincter, the greater transdiaphragmatic pressure gradient, pathological accumulation of fat and the high rate of hiatal hernia that comes with overweight will be the main factors that explain this correlation. Other factor like pathological eating habits or obesity related diseases have also an important role. In summary, it is the sum of the factors more than just one of them what would explain the pathophysiology of GERD in obese population.

Immune cell kinetics and antibody response in COVID-19 patients with low-count monoclonal B-cell lymphocytosis.

Low-count monoclonal B-cell lymphocytosis (MBLlo ) has been associated with an underlying immunodeficiency and has recently emerged as a new risk factor for severe COVID-19. Here, we investigated the kinetics of immune cell and antibody responses in blood during COVID-19 of MBLlo versus non-MBL patients. For this study, we analyzed the kinetics of immune cells in blood of 336 COVID-19 patients (74 MBLlo and 262 non-MBL), who had not been vaccinated against SARS-CoV-2, over a period of 43 weeks since the onset of infection, using high-sensitivity flow cytometry. Plasma levels of anti-SARS-CoV-2 antibodies were measured in parallel by ELISA. Overall, early after the onset of symptoms, MBLlo COVID-19 patients showed increased neutrophil, monocyte, and particularly, plasma cell (PC) counts, whereas eosinophil, dendritic cell, basophil, and lymphocyte counts were markedly decreased in blood of a variable percentage of samples, and with a tendency toward normal levels from week +5 of infection onward. Compared with non-MBL patients, MBLlo COVID-19 patients presented higher neutrophil counts, together with decreased pre-GC B-cell, dendritic cell, and innate-like T-cell counts. Higher PC levels, together with a delayed PC peak and greater plasma levels of anti-SARS-CoV-2-specific antibodies (at week +2 to week +4) were also observed in MBLlo patients. In summary, MBLlo COVID-19 patients share immune profiles previously described for patients with severe SARS-CoV-2 infection, associated with a delayed but more pronounced PC and antibody humoral response once compared with non-MBL patients.

KITD816V mutation in blood for the diagnostic screening of systemic mastocytosis and mast cell activation syndromes.

BACKGROUND: Current diagnostic algorithms for systemic mastocytosis (SM) rely on the detection of KITD816V in blood to trigger subsequent bone marrow (BM) investigations. METHODS: Here, we correlated the KITD816V mutational status of paired blood and BM samples from 368 adults diagnosed with mast cell activation syndrome (MCAS) and mastocytosis and determined the potential utility of investigating KITD816V in genomic DNA from blood-purified myeloid cell populations to increase diagnostic sensitivity. In a subset of 69 patients, we further evaluated the kinetics of the KITD816V cell burden during follow-up and its association with disease outcome. RESULTS: Our results showed a high correlation (P < .0001) between the KITD816V mutation burden in blood and BM (74% concordant samples), but with a lower mean of KITD816V-mutated cells in blood (P = .0004) and a high rate of discordant BM+ /blood- samples particularly among clonal MCAS (73%) and BM mastocytosis (51%), but also in cutaneous mastocytosis (9%), indolent SM (15%), and well-differentiated variants of indolent SM (7%). Purification of different compartments of blood-derived myeloid cells was done in 28 patients who were BM mast cell (MC)+ /blood- for KITD816V, revealing KITD816V-mutated eosinophils (56%), basophils (25%), neutrophils (29%), and/or monocytes (31%) in most (61%) patients. Prognostically, the presence of ≥3.5% KITD816V-mutated cells (P < .0001) and an unstable KITD816V mutation cell burden (P < .0001) in blood and/or BM were both associated with a significantly shortened progression-free survival (PFS). CONCLUSIONS: These results confirm the high specificity but limited sensitivity of KITD816V analysis in whole blood for the diagnostic screening of SM and other primary MCAS, which might be overcome by assessing the mutation in blood-purified myeloid cell populations.

Altered innate immune profile in blood of systemic mastocytosis patients.

Background: Mast cells (MC) from systemic mastocytosis (SM) patients release MC mediators that lead to an altered microenvironment with potential consequences on innate immune cells, such as monocytes and dendritic cells (DC). Here we investigated the distribution and functional behaviour of different populations of blood monocytes and DC among distinct diagnostic subtypes of SM. Methods: Overall, we studied 115 SM patients - 45 bone marrow mastocytosis (BMM), 61 indolent SM (ISM), 9 aggressive SM (ASM)- and 32 healthy donors (HD). Spontaneous and in vitro-stimulated cytokine production by blood monocytes, and their plasma levels, together with the distribution of different subsets of blood monocytes and DCs, were investigated. Results: SM patients showed increased plasma levels and spontaneous production by blood monocytes of IL1ß, IL6, IL8, TNFα and IL10, associated with an exhausted ability of LPS + IFNγ-stimulated blood monocytes to produce IL1ß and TGFß. SM (particularly ISM) patients also showed decreased counts of total monocytes, at the expense of intermediate monocytes and non-classical monocytes. Interestingly, while ISM and ASM patients had decreased numbers of plasmacytoid DC and myeloid DC (and their major subsets) in blood, an expansion of AXL+ DC was specifically encountered in BMM cases. Conclusion: These results demonstrate an altered distribution of blood monocytes and DC subsets in SM associated with constitutive activation of functionally impaired blood monocytes and increased plasma levels of a wide variety of inflammatory cytokines, reflecting broad activation of the innate immune response in mastocytosis.

Comprehensive Analysis of Acquired Genetic Variants and Their Prognostic Impact in Systemic Mastocytosis.

Systemic mastocytosis (SM) is a rare clonal haematopoietic stem cell disease in which activating KIT mutations (most commonly KIT D816V) are present in virtually every (>90%) adult patient at similar frequencies among non-advanced and advanced forms of SM. The KIT D816V mutation is considered the most common pathogenic driver of SM. Acquisition of this mutation early during haematopoiesis may cause multilineage involvement of haematopoiesis by KIT D816V, which has been associated with higher tumour burden and additional mutations in other genes, leading to an increased rate of transformation to advanced SM. Thus, among other mutations, alterations in around 30 genes that are also frequently mutated in other myeloid neoplasms have been reported in SM cases. From these genes, 12 (i.e., ASXL1, CBL, DNMT3A, EZH2, JAK2, KRAS, NRAS, SF3B1, RUNX1, SF3B1, SRSF2, TET2) have been recurrently reported to be mutated in SM. Because of all the above, assessment of multilineage involvement of haematopoiesis by the KIT D816V mutation, in the setting of multi-mutated haematopoiesis as revealed by a limited panel of genes (i.e., ASXL1, CBL, DNMT3A, EZH2, NRAS, RUNX1 and SRSF2) and associated with a poorer patient outcome, has become of great help to identify SM patients at higher risk of disease progression and/or poor survival who could benefit from closer follow-up and eventually also early cytoreductive treatment.

Analysis of the Emotional Exhaustion Derived From Techno-Stress in the Next Generation of Qualified Employees.

This study analyses the emotional exhaustion of students inhigher education, derived from the extremely technology-relatedstrain associated to the current COVID-19 pandemic in a conservation of resources' approach. Technostress, as source of emotional exhaustion, was investigated in a sample of 333 students in a medium size public university in Spain. Data was collected in May 2020, during the COVID lockdown. After literature review, a structural model was developed, linking technostress with emotional exhaustion. Results confirm the expected cause-effect relationships. In addition, the study reveals two mediator variables that must be considered when managing students' suffering, perceived stress and intrapersonal conflicts. This study contributes to the academic literature in the field of managing and mitigating suffering. They do so by providing both new knowledge and empirical evidence on the effects of technostress in the new generations that will soon join the working life.

Analysis of the Management of Cholelithiasis in Bariatric Surgery Patients: a Single-Center Experience.

PURPOSE: Cholelithiasis is an issue in bariatric surgery patients. The incidence of cholelithiasis is increased in morbidly obese patients. After bariatric surgery, the management maybe sometimes challenging. There is no consensus about how to deal with cholelithiasis prior to bariatric surgery. MATERIALS AND METHODS: A retrospective review from our prospectively collected bariatric surgery database. Primary bariatric procedures from 2009 to 2020 were included. Prevalence of cholelithiasis and its management prior to bariatric surgery and the incidence and management of postoperative biliary events were analyzed. RESULTS: Over 1445 patients analyzed, preoperatively cholelithiasis was found in 153 (10.58%), and 68 out of them (44.44%) were symptomatic. Seventy-six patients had a concomitant cholecystectomy. In those cases, the bariatric procedure did not show increased operative time, length of stay, morbidity, or mortality compared to the rest of primary bariatric procedures. Twelve patients (15.58%) with previous cholelithiasis and no concomitant cholecystectomy presented any kind of biliary event and required cholecystectomy. De novo cholelithiasis rate requiring cholecystectomy was 3.86%. Postoperative biliary events both in de novo and persistent cholelithiasis population did not show any difference between the type of surgery, weight loss, and other characteristics. CONCLUSIONS: Cholelithiasis was present in 10.58% of our primary bariatric surgery population. Concomitant cholecystectomy was safe in our series. Non-surgical management of asymptomatic cholelithiasis did not lead to a higher risk of postoperative biliary events. The global postoperative cholecystectomy rate was equivalent to the general population.

Outcomes of Conversion from Sleeve Gastrectomy to Roux-en-Y Gastric Bypass Due to GERD-a Retrospective Analysis of 35 Patients.

BACKGROUND: Sleeve gastrectomy (SG) has become one of the most performed bariatric procedures worldwide. Its main weaknesses are weight regain and gastroesophageal reflux disease (GERD). Conversion to Roux-en-Y gastric bypass (RYGB) is considered the gold standard to manage GERD and related symptoms. METHODS: Retrospective evaluation from a prospective bariatric surgery database of all our institution's patients converted from SG to RYGB due to GERD between January 2010 and December 2018. Clinical characteristics and workups before SG and before and after RYGB were analyzed. RESULTS: During the study period, 35 patients needed a conversion to RYGB, due to GERD or GERD-related symptoms. Mean age was 48.6 years, 85.7% were women, and mean BMI was 31.4 kg/m2. The interval between SG and RYGB was in a range 7 to 70 months (mean 33 months). All conversions were completed laparoscopically, associating a hiatoplasty in 45.7% of cases. A complete remission of symptoms was observed in 74% of patients, some improvement in 20%, and no relief in 6%. There were 3 cases of hiatal hernia persistence and 2 of recidivism. Only 1 patient presented pathological pHmetry, while moderate esophagitis was demonstrated in 2 patients. CONCLUSIONS: Conversion to RYGB was effective in almost all patients. Pathological acid exposure and hiatal hernias seem to be the main findings prior to conversion, justifying an exhaustive examination and aggressive approach to the hiatus. Due to the insufficient correlation between symptoms and findings on morphological and functional tests, actively searching for signs of GERD is advisable.

Water-soluble polymers in agriculture: xanthan gum as eco-friendly alternative to synthetics.

Water-soluble polymers (WSPs) are a versatile group of chemicals used across industries for different purposes such as thickening, stabilizing, adhesion and gelation. Synthetic polymers have tailored characteristics and are chemically homogeneous, whereas plant-derived biopolymers vary more widely in their specifications and are chemically heterogeneous. Between both sources, microbial polysaccharides are an advantageous compromise. They combine naturalness with defined material properties, precisely controlled by optimizing strain selection, fermentation operational parameters and downstream processes. The relevance of such bio-based and biodegradable materials is rising due to increasing environmental awareness of consumers and a tightening regulatory framework, causing both solid and water-soluble synthetic polymers, also termed 'microplastics', to have come under scrutiny. Xanthan gum is the most important microbial polysaccharide in terms of production volume and diversity of applications, and available as different grades with specific properties. In this review, we will focus on the applicability of xanthan gum in agriculture (drift control, encapsulation and soil improvement), considering its potential to replace traditionally used synthetic WSPs. As a spray adjuvant, xanthan gum prevents the formation of driftable fine droplets and shows particular resistance to mechanical shear. Xanthan gum as a component in encapsulated formulations modifies release properties or provides additional protection to encapsulated agents. In geotechnical engineering, soil amended with xanthan gum has proven to increase water retention, reduce water evaporation, percolation and soil erosion - topics of high relevance in the agriculture of the 21st century. Finally, hands-on formulation tips are provided to facilitate exploiting the full potential of xanthan gum in diverse agricultural applications and thus providing sustainable solutions.

Volatile Organic Compound Chamber: A Novel Technology for Microbiological Volatile Interaction Assays.

The interest in the study of microbiological interactions mediated by volatile organic compounds (VOCs) has steadily increased in the last few years. Nevertheless, most assays still rely on the use of non-specific materials. We present a new tool, the volatile organic compound chamber (VOC chamber), specifically designed to perform these experiments. The novel devices were tested using four Trichoderma strains against Fusarium oxysporum and Rhizoctonia solani. We demonstrate that VOC chambers provide higher sensitivity and selectivity between treatments and higher homogeneity of results than the traditional method. VOC chambers are also able to test both vented and non-vented conditions. We prove that ventilation plays a very important role regarding volatile interactions, up to the point that some growth-inhibitory effects observed in closed environments switch to promoting ones when tested in vented conditions. This promoting activity seems to be related to the accumulation of squalene by T. harzianum. The VOC chambers proved to be an easy, homogeneous, flexible, and repeatable method, able to better select microorganisms with high biocontrol activity and to guide the future identification of new bioactive VOCs and their role in microbial interactions.

Overwhelmed by Technostress? Sensitive Archetypes and Effects in Times of Forced Digitalization.

This paper explores technostress and its dimensions, assessing the relationship with possible negative effects in the individual, social and professional sphere. The study uses a self-reported approach of undergraduate students in Spain (n = 337), forced to follow their academic life by using technology comprehensively because of social distancing, as a public health action necessary to reduce the spread of COVID-19. The analysis, based on the exploration of a system of archetypes of the use of social networks, presents insights into contemporary technostress management as a new approach that can suppose opportunities for the optimization of prevention plans. Pearson's correlation coefficients and structural equation modeling based on partial least squares (SEM-PLS) were the methods used for achieving the goals. The results reveal valid and reliable measures where technostress has a high impact on the individual sphere of students and there is a significant relationship between the type of user and techno-anxiety. The conclusions point to the imperative for developing a deeper understanding of technostress by archetypes, in both a higher education context (as antecedent) and the world of work, in an irreversible move towards a digital economy.

Seeking an Initial-Weight-Independent Metric in a Mediterranean Cohort of Gastric Bypass Patients: the %AWL Revisited.

BACKGROUND: Most relative weight-loss metrics follow the formula "Weight loss(%) = 100 · (Initial BMI - Final BMI) / (Initial BMI-a)," where a is the reference point that defines the metric. The percentage of total weight loss (%TWL, a = 0) and percentage of excess weight loss (%EWL, a = 25) are influenced by a patient's initial weight. Recently, the percentage of alterable weight loss metric (%AWL, a = 13) has been reported to produce initial-weight-independent outcomes. OBJECTIVES: This study aimed to replicate the methodology used for %AWL determination in a Mediterranean cohort of bariatric patients. SETTINGS: Multicenter study in 10 large hospitals in Spain. METHODS: Two large prospective databases were retrospectively searched for all primary laparoscopic gastric bypass patients with 2 years of follow-up. Outcomes at nadir were expressed and analyzed with 26 different metrics (a from 0 to 25), looking for the metric whose outcomes produced (1) the lowest coefficient of variation, (2) no differences between initially lighter and heavier patients, and (3) no correlation with patients' initial BMI. RESULTS: A cohort of 1793 patients was stratified into 4 gender-age groups: younger women (YW, n = 733), older women (OW, n = 674), younger men (YM, n = 197), and older men (OM, n = 189). The calculations suggested an optimal reference point of 18 kg/m2, defining a new metric (percentage of Mediterranean alterable weight loss, %MAWL). When %TWL, %EWL, %AWL, and %MAWL were tested on the whole sample, only %MAWL produced initial-weight-independent results. CONCLUSIONS: In our Mediterranean cohort of patients, a reference point of 18 (and not 13) yielded initial-weight-independent outcomes.

Effect of Cry Toxins on Xylotrechus arvicola (Coleoptera: Cerambycidae) Larvae.

The beetle Xylotrechus arvicola is a destructive pest in vineyards (Vitis vinifera) in the main wine-producing areas of the Iberian Peninsula. X. arvicola larvae bore into the grapevine wood-making galleries, thus damaging the plant both directly and indirectly; the latter through the proliferation of wood fungi, which can invade the inside of the plant, decreasing the quality and quantity of its production. The susceptibility of X. arvicola larvae to five coleopteran toxic Cry proteins (Cry1B, Cry1I, Cry3A, Cry7A, and Cry23/37) was evaluated under laboratory conditions in order to deepen the knowledge of the effect of these proteins on this insect throughout its biological development. Cry7Ab and Cry1Ba were the most effective in controlling X. arvicola larvae due to the significant reduction in larvae survival (32.9 and 25.9 days, respectively), and by causing serious alterations in the larvae during the remaining months of their development. The developmental stage of the prepupal and pupal stages was not affected by the previous ingestion of Cry proteins. The Cry proteins tested could be applied to control X. arvicola larvae since they were able to kill them and cause serious alterations in the larvae during the remaining months of development that followed. The data presented suggest that these Cry proteins can be used as bioinsecticides against the larvae of this insect, applying them only at the moment when the larvae hatch from the egg outside the grapevine wood (this would only be useful and justified if the economic threshold is exceeded) in order to avoid the rapid evolution of resistance against these toxins since not all of the larvae were killed and thus increase vine wood protection.

Work-Life Balance in Great Companies and Pending Issues for Engaging New Generations at Work.

The changing nature of employment and work causes new demands in society, such as work-life balance, that has emerged in labor relations as an important aspect of a healthy work environment. In this context, Best Companies to Work for are a reference in caring for their staff, and it is well known that new generations-that frequently use the Internet to be informed-are making their decisions as job seekers by checking and comparing corporate websites. In order to learn from the best companies, but also to discover what could be improved by identifying the gaps, this study observes the current work-life balance practices in the last Best Companies to Work for awarded by Fortune. The main contribution of this work is the development of a weighted index for benchmarking purposes considering the preferences of new generations at work. The study demonstrates that the best companies still report low levels of work-life balance information. The main implication drawn from the study, due the requirements of new generations at work and the rapidly emerging field of e-recruiting, is the need for human resource departments to fit work and personal life in a fluid way, while maintaining a healthy balance. It is also recommended for companies to improve their disclosure of work-life practices on line for attracting talent from Millennials and Generation Z.

Well-Being Lessons for Improving Charities' Online Recruitment.

In an increasingly competitive context, attracting and retaining the best employees are a real preoccupation and a big challenge for organizations. Online recruitment (OR) is a growing trend, and corporate websites are an important instrument for talent attraction, but academic research on this topic is still scarce, especially in the voluntary sector. To shed light on the topic, this study examines and compares the 100 best companies to work for, published by Fortune, and the 100 largest charities, reported by Forbes. The comparative study focuses the attention and quantifies the web section devoted to careers, concretely information related to goods practices affecting the workers well-being. The results indicated, as essential in the OR process of charities, to understand the relevance of their web content because that affects the intentions of potential applicants. The work concludes that benchmarking efforts can be helpful for increasing the charities' attractiveness in the labor market in the near future.

Insecticidal Properties of Ocimum basilicum and Cymbopogon winterianus against Acanthoscelides obtectus, Insect Pest of the Common Bean (Phaseolus vulgaris, L.).

The bean weevil, Acanthoscelides obtectus Say (Coleoptera: Chrysomelidae: Bruchinae), causes severe post-harvest losses in the common bean, Phaseolus vulgaris L. The control of this insect is still poor and involves the use of conventional insecticides. There is an increasing demand in the search for new active substances and products for pest control towards reduction of adverse effects on human health and the environment. The protection of grains with alternative products, such as essential oils, is a possible alternative to meet the needs described above. Therefore, this investigation evaluated the applications of basil, Ocimum basilicum, and citronella, Cymbopogon winterianus, essential oils for A. obtectus control. These essential oils significantly reduced the bean weight losses and the number of beans damaged by A. obtectus at higher doses than 60 or 120 µL/sample. The number of holes per bean did not differ between the doses of basil essential oil, not even at the dose of 60 µL, while it was higher at 120 µL, probably due to a lower capacity of movement of the insects treated with this dose and/or the oil's direct or indirect effects on the insects. Basil and citronella oils exhibited similar patterns of insecticidal activity over the insect, both directly in adult insects or indirectly over bean seeds. These essential oils affected the development of A. obtectus since the greatest doses applied on beans decreased the emergence of the bean weevil. The results prove the insecticidal capacity of the tested essential oils and hence their potential as active substances against A. obtectus in environmentally low risk pest control strategies. Supplementary trials should be conducted under real storage conditions.

One-step nucleic acid amplification for intraoperative analysis of sentinel lymph node in papillary thyroid carcinoma.

Objective Lymphadenectomy in papillary thyroid carcinoma (PTC) is controversial. It is indicated whenever metastases have been proven before or during surgery and as a prophylactic treatment in high-risk patients. However, 30-50% of cN0 patients become pN1 postoperatively. In PTC, selective-sentinel-lymph-node-biopsy (SLNB) with conventional intraoperative analysis is 8% false negative. One-step nucleic acid amplification (OSNA) is a molecular technique which allows real-time detection of mRNA encoding for cytokeratin 19. OSNA has been introduced in intraoperative analysis of several tumors to reduce false-negative rates and distinguish micrometastasis from macrometastasis. Our objective was to evaluate the impact of the introduction of OSNA in the intraoperative evaluation of the sentinel node (SN) in PTC. Design We analyzed a series of 35 patients subjected to SLNB. Methods All the dissected nodes, SN and non-SN, were evaluated with OSNA and cytology. Results We obtained a total of 110 SN. SLNB proved positive in 14 patients (40%) with cytology and in 23 (65.7%) with OSNA (P < 0.001). In the 29 patients with subsequent lymphadenectomy we obtained 360 lymph nodes ((52 positive in cytology (14.4%) and 107 in OSNA (29.7%)). Lymphadenectomy proved positive in 16 patients according to cytology (55%) and in 24 according to OSNA (83%) (P = 0002). The majority of patients with micrometastasis in SN showed only micrometastasis in lymphadenectomy. Conclusions The present study shows selective-sentinel-lymph-node-biopsy with one-step nucleic acid amplification technique to be feasible in papillary thyroid carcinoma. The quantitative nature of one-step nucleic acid amplification paves the way toward a more personalized surgical approach, limiting lymphadenectomy to patients with intraoperative evidence of macrometastasis in the sentinel node.

Maintenance and assessment of cell viability in formulation of non-sporulating bacterial inoculants.

The application of beneficial, plant-associated microorganisms is a sustainable approach to improving crop performance in agriculture. However, microbial inoculants are often susceptible to prolonged periods of storage and deleterious environmental factors, which negatively impact their viability and ultimately limit efficacy in the field. This particularly concerns non-sporulating bacteria. To overcome this challenge, the availability of protective formulations is crucial. Numerous parameters influence the viability of microbial cells, with drying procedures generally being among the most critical ones. Thus, technological advances to attenuate the desiccation stress imposed on living cells are key to successful formulation development. In this review, we discuss the core aspects important to consider when aiming at high cell viability of non-sporulating bacteria to be applied as microbial inoculants in agriculture. We elaborate the suitability of commonly applied drying methods (freeze-drying, vacuum-drying, spray-drying, fluidized bed-drying, air-drying) and potential measures to prevent cell damage from desiccation (externally applied protectants, stress pre-conditioning, triggering of exopolysaccharide secretion, 'helper' strains). Furthermore, we point out methods for assessing bacterial viability, such as colony counting, spectrophotometry, microcalorimetry, flow cytometry and viability qPCR. Choosing appropriate technologies for maintenance of cell viability and evaluation thereof will render formulation development more efficient. This in turn will aid in utilizing the vast potential of promising, plant beneficial bacteria as sustainable alternatives to standard agrochemicals.