Effect of culture conditions on the performance of lignocellulose-degrading synthetic microbial consortia

In this study, we examined a synthetic microbial consortium, composed of two selected bacteria, i.e., Citrobacter freundii so4 and Sphingobacterium multivorum w15, next to the fungus Coniochaeta sp. 2T2.1, with respect to their fate and roles in the degradation of wheat straw (WS). A special focus was placed on the effects of pH (7.2, 6.2, or 5.2), temperature (25 versus 28 °C), and shaking speed (60 versus 180 rpm). Coniochaeta sp. 2T2.1 consistently had a key role in the degradation process, with the two bacteria having additional roles. Whereas temperature exerted only minor effects on the degradation, pH and shaking speed were key determinants of both organismal growth and WS degradation levels. In detail, the three-partner degrader consortium showed significantly higher WS degradation values at pH 6.2 and 5.2 than at pH 7.2. Moreover, the two bacteria revealed up to tenfold enhanced final cell densities (ranging from log8.0 to log9.0 colony forming unit (CFU)/mL) in the presence of Coniochaeta sp. 2T2.1 than when growing alone or in a bacterial bi-culture, regardless of pH range or shaking speed. Conversely, at 180 rpm, fungal growth was clearly suppressed by the presence of the bacteria at pH 5.2 and pH 6.2, but not at pH 7.2. In contrast, at 60 rpm, the presence of the bacteria fostered fungal growth. In these latter cultures, oxygen levels were significantly lowered as compared to the maximal levels found at 180 rpm (about 5.67 mg/L, ~ 62% of saturation). Conspicuous effects on biomass appearance pointed to a fungal biofilm–modulating role of the bacteria.Key points• Coniochaeta sp. 2T2.1 has a key role in wheat straw (WS) degradation.• Bacterial impact shifts when conditions change.• pH and shaking speed are key drivers of the growth dynamics and WS degradation.

Impact of Plasmatic Progesterone on the Day of Frozen Embryo Transfer in Hormone-induced Cycles

Objective To establish a relationship between serum progesterone values on the day of frozen blastocyst transfer in hormone-replaced cycles with the probability of pregnancy, miscarriage or delivery. Methods This was an ambispective observational study including all frozen-thawed embryo transfer cycles performed at our department following in vitro fecundation from May 2018 to June 2019. The outcomes evaluated were β human chorionic gonadotropin (β-hCG)-positive pregnancy and delivery. Groups were compared according to the level of serum progesterone on the day of embryo transfer: the 1st quartile of progesterone was compared against the other quartiles and then the 2nd and 3rd quartiles against the 4th quartile. Results A total of 140 transfers were included in the analysis: 87 with β-HCG > 10 IU/L (62%), of which 50 (36%) delivered and 37 had a miscarriage (42%). Women with lower progesterone levels (< 10.7ng/mL) had a trend toward higher β-HCG-positive (72 versus 59%; p > 0.05), lower delivery (26 versus 39%; p > 0.05) and higher miscarriage rates (64 versus 33%; p < 0.01). Comparing the middle quartiles (P25–50) with those above percentiles 75, the rate of pregnancy was similar (60 versus 57%; p > 0.05), although there was a trend toward a higher number of deliveries (43 versus 31%; p > 0.05) and a lower number of miscarriages (28 versus 45%; p > 0.05). These differences were not statistically significant. Conclusion There were no differences in pregnancy and delivery rates related with the progesterone level when measured in the transfer day. The miscarriage rate was higher in the 1st quartile group.

Cerebrospinal fluid hemoglobin levels as markers of blood contamination: relevance for α-synuclein measurement

Objectives Cerebrospinal fluid α-synuclein (CSF α-syn) represents a possible biomarker in Parkinson’s disease (PD) diagnosis. CSF blood contamination can introduce a bias in α-syn measurement. To date, CSF samples with a red blood cells (RBC) count >50 RBC × 106/L or haemoglobin (Hb) concentration >200 μg/L are excluded from biomarker studies. However, investigations for defining reliable cut-off values are missing. Methods We evaluated the effect of blood contamination on CSF α-syn measurement by a systematic approach in a cohort of 42 patients with different neurological conditions who underwent lumbar puncture (LP) for diagnostic reasons. CSF samples were spiked with whole blood and serially diluted to 800, 400, 200, 100, 75, 50, 25, 5, 0 RBC × 106/L. CSF α-syn and Hb levels were measured by ELISA. Results In neat CSF, the average concentration of α-syn was 1,936 ± 636 ng/L. This value increased gradually in spiked CSF samples, up to 4,817 ± 1,456 ng/L (+149% α-syn variation) in samples with 800 RBC × 106/L. We established different cut-offs for discriminating samples with α-syn level above 5, 10, and 20% variation, corresponding to a Hb (RBC) concentration of 1,569 μg/L (37 RBC × 106/L), 2,082 μg/L (62 RBC × 106/L), and 3,118 μg/L (87 RBC × 106/L), respectively. Conclusions Our data show the high impact of CSF blood contamination on CSF α-syn levels, highlighting the measurement of Hb concentration as mandatory when assessing CSF α-syn. The thresholds we calculated are useful to classify CSF samples for blood contamination, considering as reliable only those showing a Hb concentration <1,569 μg/L.

Molecularly Imprinted Polyscopoletin for the Electrochemical Detection of the Chronic Disease Marker Lysozyme

Herein we report the electropolymerization of a scopoletin based molecularly imprinted polymer (MIP) for the detection of lysozyme (Lyz), an enzymatic marker of several diseases in mammalian species. Two different approaches have been used for the imprinting of lysozyme based, respectively, on the use of a monomer-template mixture and on the covalent immobilization of the enzyme prior to polymer synthesis. In the latter case, a multi-step protocol has been exploited with preliminary functionalization of gold electrode with amino groups, via 4-aminothiophenol, followed by reaction with glutaraldehyde, to provide a suitable linker for lysozyme. Each step of surface electrode modification has been followed by cyclic voltammetry and electrochemical impedance spectroscopy, which has been also employed to test the electrochemical responses of the developed MIP. The sensors show good selectivity to Lyz and detect the enzyme at concentrations up to 292 mg/L (20 μM), but with different performances, depending on the used imprinting approach. An imprinting factor equal to 7.1 and 2.5 and a limit of detection of 0.9 mg/L (62 nM) and 2.1 mg/L (141 nM) have been estimated for MIPs prepared with and without enzyme immobilization, respectively. Competitive rebinding experiment results show that this sensing material is selective for Lyz determination. Tests were performed using synthetic saliva to evaluate the potential application of the sensors in real matrices for clinical purposes.

Effect of iodine-containing antiseptics on urine iodine levels of surgical staff after iodization

BackgroundThe routine use of topical iodinated antiseptic could be a possible cause of iodine contamination. This study was aimed to determine urinary iodine status of operation-room staff who routinely use iodine-containing antiseptics for cleansing of the hands after salt iodization.Materials and methodsThe study included 40 operation-room staff who use surgical hand-scrub solutions. Participants applied an iodized brush for a minimum of three times a day on weekdays and were iodine-free on the weekends. Morning urine samples from all volunteers to analyze the urinary iodine concentration (UIC) twice a week. Modified microplate method of Sandell-Kolthoff reaction used to measure UIC.ResultsThe UICs were significantly higher on Friday (median 194 μg/L (70–396 μg/L)) compared to the Monday concentrations (median 125 μg/L (62–264 μg/L), p < 0.001). Mild iodine deficiency occurred in 32.5% of the subjects on Monday, in 5% on Friday. On Monday, there were no subjects with UIC > 300 μg/L, but on Friday, 7.5% of the subjects had UIC > 300 μg/L.ConclusionDespite both the use of iodized antiseptic solution and mandatory iodization, operation-room staff had only median iodine levels with low risk of iodine intoxication.

A Synthetic Approach for Biosynthesis of Miquelianin and Scutellarin A in Escherichia coli

Grapevine (Vitis vinifera) glycucuronosyltransferase (VvGT5) specifically catalyzes flavonol-3-O-glucuronosylation and the blue flowers of Veronica persica (Lamiales, Scrophulariaceae) uridine diphosphate (UDP)-dependent glycosyltransferase (UGT88D8) as flavonoid 7-O-specific glucuronosyltransferases, were chosen, codon optimized, and employed to synthesize the high valued flavonoids glucuronoids, miquelianin and scutellarin A in Escherichia coli. A single vector system was constructed to overexpress entire UDP-glucuronic acid biosynthesis pathway genes, along with a glucokinase gene in Escherichia coli BL21 (DE3). The newly generated E. coli BL21 (DE3) piBR181-glk.pgm2.galU.ugd.UGT88D8 strain produced 12 mg/L (28 µmol/L) of scutellarin A from apigenin, representing only 14% of maximum conversion percentage. Similarly, the strain E. coli BL21 (DE3) piBR181-glk.pgm2.galU.ugd.VvGT5 produced 30 mg/L (62 µmol/L) of miquelianin, representing a 31% conversion of quercetin. This production profile is a good starting point for further host engineering, and for production of respective compounds.

Azacitidine Response Prediction in MDS Patients with NGS Data Using a Computational Biology Modeling (CBM) Based Clinical Decision Support System

BACKGROUND: Azacitidine (AZA) is currently a drug of choice for most of high-risk MDS patients. However, only 40-50% of MDS patients achieve clinical improvement with AZA. There is a need for a predictive clinical decision support tool that can identify MDS patients with higher or lower likelihood of AZA response. Ideally, patients with no chance of response would be spared of life-threatening toxicities and expense; while patients with high chance for response would receive maximized treatment. AIM: The objective of this study was to predict response to AZA in a cohort of intermediate- and high-risk MDS patients using CBM approach in retrospective blinded manner. METHODS: We analyzed the clinical and genomic (NGS, cytogenetics and FISH) data for a cohort of 48 Int-2 and high risk MDS patients treated with AZA for median of 12 (4-34) cycles. Median age was 70.4 years, M:F ratio 1:1. MDS subtypes: EB-2 (35.4%, N=17), EB-1 (39.6%, N=19), MDS/AML (18.6%, N=9), CMML1/2 (4.2%, N=2), and RARS (2.1%, N=1). Median IPSS-R was 5 (3-10), cytogenetic score was 1. Median BM blasts were 10% (0.88-43.12), Hemoglobin 91g/L (62-145), ANC 1.24x109/mL (0.09-11.64), Platelets 84x109/mL (2-576). Patients were treated by AZA until progression to AML. Median AZA cycles was 14. Median overall survival (OS) on therapy was 24.2 months (4.4-61.6). Median progression free survival (PFS) was 15.9 months (4.0-61.6). Clinical responders were defined by IWG2006 criteria. Overall response rate (ORR) was 60.4% (CR/PR: 18 of 48 patients, stable disease with hematology improvement (SD-HI): 11/48). While 29 of 48 (60.4%) patients progressed to AML following the AZA therapy; 5 patients (10.4%) were primarily AZA-resistant. CBM for each MDS patient was created utilizing genomic data to create a predictive workflow (Cellworks Group) complemented with digital mechanistic model of AZA and other FDA-approved drugs. Drugs were modeled by programming their mechanism of action on pathways and simulated individually and in combination. A disease inhibition score (DIS) characterized the drug impact to which malignant phenotypes was inhibited. For AZA non-responder profiles, unique combinations were selected that reduced DIS. RESULTS: CBM cohort analysis performed on 37 out of 48 patients predicted 20 clinical responders (54.05%) and 17 clinical non-responders (45.95%). CBM accurately predicted the clinical outcomes of 14 out of 20 responders and 17 out of 17 non-responders with overall accuracy 83.78%. Sensitivity of identifying a responder is 70% while non-responders are called with 100% specificity. The CBM identified AZA based combination in 17 patients who did not respond to AZA monotherapy: AZA+Lenalidomide (N=6), AZA+Dasatinib (1), AZA+Ruxolitinib (2), AZA+Sorafenib (1), and AZA+Venetoclax (7). In the patient AZA014 (EB1 with isolated 5q-) who underwent AZA for 17 cycles and responded with mCR (MLD) without HI, the CBM predicted Lenalidomide sensitivity. Following the treatment with Lenalidomide the patient entered a long lasting CR (> 3 years). Digital droplet ddPCR technology during patient follow-up confirmed loss of the clone characterized by TP53(p.Thre377Pro) mutation upon Lenalidomide therapy. In contrast, ddPCR of AZA048 utilized SF3B1 (p.Lys700Glu) mutation tracking during patient follow-up that predicted the relapse by 8 months. CONCLUSION: The CBM prediction of AZA clinical response in newly diagnosed, higher risk MDS patients showed high predictive accuracy of AZA resistant patients. The study validates the approach to a priori predict response and identify the right therapy option for the patient and could be used to establish criteria for precision enrollment in drug development trials. In addition, analysis uncovered possible mechanisms for AZA resistance that could be targeted to induce response. Clonal architecture is trackable using ddPCR technology providing time for additional NGS analysis to target the progressing clone. Ultimately, improving patient selection and mutation tracking may avoid unnecessary chemotherapy toxicity and reduce health care expenses. Disclosures Abbasi: Cell Works Group Inc.: Employment. Singh:Cellworks Research India Private Limited: Employment. Ullal:Cellworks Research India Private Limited: Employment. Narayanabhatia:Cellworks Research India Private Limited: Employment. Alam:Cellworks Research India Private Limited: Employment. Roy:Cellworks Research India Private Limited: Employment. Choudhary:Cellworks Research India Private Limited: Employment. Vali:Cell Works Group Inc.: Employment. Cogle:Celgene: Other: Steering Committee Member of Connect MDS/AML Registry.

An ethnobotanical study of medicinal plants used in Zacatecas state, Mexico

Despite the fact that Mexico has vast biocultural biodiversity, there are numerous regions where the traditional medicinal use of plants has not yet been studied. We aimed to document, analyze quantitatively, and preserve medicinal plant knowledge among local people living in over 40 communities in the state of Zacatecas. Ethnobotanical information was collected by semistructured interviews with 132 informants. Data were analyzed using standard quantitative indices such as relative frequency of citation, family importance value, cultural importance index, and informant consensus factor. We recorded 168 medicinal plant taxa belonging to 151 genera and 69 botanical families and used to treat 99 health disorders. The most medicinally important plant families were Asteraceae (20 species), followed by Fabaceae and Lamiaceae (12 species) and Cactaceae (five species). The most culturally important species was <em>Matricaria chamomilla</em> L., mentioned 140 times, followed by <em>Arnica montana</em> L. (62 times) and <em>Artemisia ludoviciana</em> Nutt (48 times). The highest consensus for use was for diseases of the reproductive system. The type of disorder for which there was the highest number of references for use (389; 25% of all uses) and plant species (67) were diseases of the digestive and gastrointestinal system. The present study represents the first quantitative medical-ethnobotanical documentation and analysis of the traditional use of medicinal plants in Zacatecas state. Despite the semiarid climate, this region is botanically highly diverse, and its flora have versatile medicinal uses.

Analysis of Clinical Characterization of CMML.

Abstract 4853 Chronic myelomonocytic leukemia(CMML) was incorporated into the FBA classification of myelodysplastic syndromes(MDS) because dysplastic changes are commonly found in blood and bone marrow cells of patients with this order. However, sometimes doctors misdiagnose it because it may be distinguishable by only the presence of monocytosis(>1.0×109/L) in the blood.CMML is always a rather controversial type of MDS, some of patients present charaterication of myeloproliferation. In a further step, the WHO classification now includes CMML in a category of mixed myeloproliferative/myelodysplastic disorders, together with atypical CML and JMML,and proposed to separate CMML into CMML I and CMML II [Harris et al. Journal of Clinical Oncology,1999; Bennett International Journal of Hematology,2000]. Prognosis is also extremely variable in CMML.The median survival was about 19 months in 288 CMML patients included in Düsseldorf MDS Registry. There was no significant difference in median survival of MPD-CMML and MDS-CMML[Nosstinger et al. Leukemia Researsh.2001]. Germing reported the median of CMML patients who developed AML(18%) was 14 months,as compared to 20 months for patients who did not develop AML. [Germing et al. Leukemia and Lymphoma. 2004].In 2002, the M.D.Anderson Prognostic Score(MDAPS) using lymphocyte counts, hemoglobin level, medullary blast count, and presence of immature myeloid precursors in blood was developed by Onida[Onida et al. Blood. 2002]. Here we analyzed the clinical characterization of CMML in our hospital. 16 cases of CMML diagnosed according to the criteria of WHO classification were retrospectively analyzed. The median age was 51 years,and female/male was 11/5.Most of patients were median or older men with splenomegaly(37%), figure(30%), bone pain(25%), hepatomegaly(13%). White blood counts varyied from hypoleukocytosis to normal to hyperleukocytosis, median count was 27×109/L (1.3-74×109/L), median platelet count was 97×109/L(12-576×109/L),median hemoglobin count was 91g/l(62-138 g/l), median monocyte count was 4.6×109/L(2-14.5×109/L), median lymphocyte count was 4.4×109/L(1.0-33.8×109/L), LDH was elevated. Bone marrow was obviously active with dyshaematopoiesis, median blast cells of granulocyte were 8.5%(1%-18%), immature monocyte was 0-2.5%. 44% of patients had eosinophilic cells and/or basophil cells increase in bone marrow, and decrease in NAP, median score was 13(0-62). Immunophenotypic feature was CD13+CD14+CD56+,and FISH: BCR-ABL(-).The clinical course of CMML could remain stable for many years only low dose chemotherapy or without any treatment. On the other hand, there were patients with rapidly progressive disease. The Median survival was 20 months. CMML-I cases average survival was 50+ months, CMML-II was 18+ months≤ MDAPS showed low risk group had a average survival of 60+ months, intermediate group 1 and 2 was 30+ months and 17+ months,high risk patients with a average survival of only 1+ months. Only one case transformed into M4 as late as 3+ years after diagnosis of CMML(6%). In conclusion, the new criteria of WHO classification presents clinical feature of CMML better, and it can provide more effective prognostic parameters for risk assessment with MDAPS. Disclosures No relevant conflicts of interest to declare.