Bacillus genus has been used in horticultural crops as a biocontrol agent against insect pests, microbial phytopathogens, and plant growth-promoting bacteria (PGPB), representing an alternative to agrochemicals. In particular, B. cereus (Bc) and B. thuringiensis (Bt) have been studied for their fungicidal and insecticidal activities. However, their use as biofertilizer formulations and biocontrol agents against phytopathogenic bacteria is limited.Xanthomonas euvesicatoria (Xe) in greenhouse-grown chili peppers.To evaluate Bc and Bt formulations as PGPB and biocontrol agents against the bacterial spot agent in vitro antagonism assays were performed by confronting Bc and Bt isolate formulations against Xe isolates in direct (foliage) and indirect (resistance induction) phytopathogen biocontrol assays on pepper plants, which were grown under controlled conditions for 15 d after formulations treatment.Bc and Bt isolates obtained from soil samples were identified and characterized using conventional biochemical and multiplex PCR identification methods. Bioassays to determine Bc and Bt isolates potential as PGPB were evaluated on chili pepper seedlings in seedbeds. In addition, formulations based on Bc (F-BC26 and F-BC08) and Bt (F-BT24) strains were assessed as biofertilizers on pepper, under controlled conditions. Furthermore, in vitro bioassays demonstrated the bactericidal effect of Bc and Bt strains against Xe isolates. Furthermore, assays showed significant plant protection by F-BC26, F-BC08, and F-BT24 formulated strains against Xe.Isolates were identified as Bc and Bt. Formulations significantly improved pepper growth in seedbeds and pots, whereas Xanthomonas euvesicatoria.Results indicated that F-BT24 and F-BC26 isolates formulations promoted pepper growth and protected it against  Capsicum annuum L.) is one of the most cultivated and consumed vegetables worldwide, after potatoes and tomatoes (Xanthomonas euvesicatoria (Xe) is the bacterial spot causal agent and may lead to crop yield reduction by up to 66%, with losses of up to $7,500 US dollars per hectare  , and B. velezensis colonize plant roots, producing exopolysaccharides, siderophores, or phytohormones, as well as solubilize phosphorus or fix nitrogen  . Bacillunitrogen . Bacillual crops . StudiesB. thuringiensis has been shown as PGPB in cabbage, pepper, lettuce, and tomato crops, with significant increases in several vegetative development parameters  in plants and controlling several microbial diseases  with Bt. Moreover, B. cereus BS107 to pepper plants in the control of X. axonopodis pv. Vesicatoria, whereas X. campestris pv. vesicatoria in tomato seedlings after B. subtilis CBR05 application. Despite these benefits, limited formulated products based on B. cereus are available as biofertilizers , unless otherwise specified. Molecular biology assay reagents were obtained from Promega Corp. . Field experiments were approved by the Fundación Produce, Chihuahua, A.C.Soil samples were collected from 30 chili pepper commercial lots from pepper producers in Meoqui, Chihuahua, MX , who are current and former members of the Fundación Produce Chihuahua, A.C. Sampling was performed by selecting five diagonal points per lot, and soil was collected at a depth of 20 cm at each point . Samples were then placed in new zip-lock plastic bags, labeled, placed in a cooler, and transported to the Applied Microbiology, Plant Pathology, and Postharvest Physiology Laboratory (MAFFP) of the Autonomous University of Chihuahua (UACH), where they were kept at 4 °C, until use.Bacillus strains isolation was performed according to the method adapted by 6 , KOH tests, and Schaeffer-Fulton stain were also performed to determine endospores presence , 1.5 µL of DNA at 5 ng/µL, and 0.2 µL of 5 U/µL Taq DNA polymerase. PCR reactions were performed using a thermocycler . The amplification program consisted of an initial denaturation cycle at 94 °C for 5 min, 30 denaturation cycles at 94 °C for 30 s, and 63 °C for 30 s, an extension at 72 °C for 30 s, and a final extension cycle at 72 °C for 5 min, followed by an initial denaturation cycle at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 5 min, annealing at 64 °C for 1 min, extension at 72 °C for 1 min, and a final extension cycle at 72 °C for 7 min . We used 25 µL samples in the PCR reaction mix, including 15.5 µL of sterile water, 5 µL of 5X PCR buffer, 1.5 µL of 25 mM MgClor 7 min . ResultsBacillus activity as PGPB in pepper plants was evaluated in two stages, under greenhouse conditions (27 °C ± 2 °C and 75% relative humidity (RH)). In the first experiment, we evaluated pepper seedlings growth promotion (seedling emergence) by 22 Bacillus strains. In a second experiment, three formulations were prepared with selected isolated identified and coded as F-BC26 for B. cereus strain CBC26, F-BC08 for B. cereus strain CBC08, and F-BT24 for B. thuringiensis strain CBT24. They were grown in 100 mL of tryptone soy broth (TSB) at room temperature for 72 h, under continuous shaking at 120 rpm. After incubation and sporulation, cultures were placed in a water bath at 90 °C for 10 min to eliminate other non-forming spore bacteria, present as contaminants. Next, the culture was centrifuged at 9,000 rpm and suspended in 10 mL of 1X phosphate buffered saline (PBS) solution (Fisher Scientific), which was inoculated in one liter of TSB and incubated at room temperature for 24 h at 120 rpm.2 and 300 mL of isopropyl alcohol. The final volume was processed in the spray dryer, with a turbine pressure of 1.5 bar, an inlet flow of 55 mL/min at 200 °C, and an outlet temperature of 80 °C ± 3 °C, after which microcapsules were prepared by spray-drying . The bacterial mixture as consortium was not included as treatment because, in previous experiments, they did not show differences from their individual effect (data not shown).Capsicum annuum var M, Southern Star Seeds S. de R.L. de C.V., Mexico City). There were grown in 20-cavity polystyrene trays filled with sterile horticultural perlite (1 h at 120 °C and 15 lb/in2 pressure). Seedlings were watered every third day with a nutrient solution composed of 5.40 milliequivalents/liter (mEq/L) KNO3,4.40 mEq/L NH4NO3, 2.60 mEq/L Ca(H2PO4)2, 1.00 mEq/L MgSO4, 8 ppm Fe (EDTA-Fe 6%), 1 ppm boric acid, and 12 ppm Fetrilon Combi®  adjusted to pH 5.5 and electrical conductivity (EC) of 1.3 milliSiemens (mS)/cm /cm .Bacillus suspensions were prepared by cultivating bacteria in NB for 72 h at 28 °C, after which they were centrifuged at 7000 rpm for 10 min at 4 °C. Next, the supernatant was removed, and the resulting pellet was suspended in a sterile saline solution 0.85%. Suspensions were adjusted to 1 ×108 CFU/mL, corresponding to an optical density (OD) of 0.4 at 600 nm (Bacillus isolates or B. subtilis QST713 (Serenade®) suspensions at 1 × 108 CFU/mL. After 40 d inoculation, plant height, stem diameter, root length, leaf number, leaf area (Canopeo app (https://canopeoapp.com)), and leaves, stem, and root dry biomass were determined. The Dickson Quality Index (DQI) was calculated using the Twenty-two t 600 nm , and app2 pressure) . Treatments were inoculated five times at 7-d intervals, immersing trays (drench) with the microgranular formulations F-BC26, F-BC08, and F-BT24 (1 × 108 CFU/mL) or B. subtillis  (1 × 107 CFU/mL). Seedlings were watered every third day with a nutrient solution containing 10% N, 8% P2O5, 18% K2O, 2.5% S, 1.8% Mg, 5.9% Ca, 0.1% Fe, 0.002% B, 0.01% Zn, 0.0002% Cu, 0.0002% Mn  and adjusted to pH 5.5 and 1.5 mS EC. On day 40 after inoculation, plant height, stem diameter, root length, leaf area (Canopeo app), and fresh and dry biomass of leaves, stems, and roots were determined.In the seedling emergence assay, we used 10-d growth Jalapeño pepper seedlings in polystyrene trays with 20 cavities, filled with sterile peat moss (1 h at 120 °C and 15 lb/in8 CFU/mL), 50 mL of BactoRacine-B® (1 × 107 CFU/mL), or 50 mL of nutrient solution were applied to each plant stem base. All plants were watered every third day with nutrient solution . After 85 d of first inoculation (transplant time), plant height, stem diameter, root length, leaf area, the number of leaves, and fresh and dry biomass of leaves, stems, and roots were determined. Carotenoids, chlorophyll “a”, and chlorophyll “b” content were also determined . Formulations were applied five times at intervals of 10 d from the transplant time. To achieve this, 50 mL of formulated suspensions . In addition, B. subtilis QST 713 (Serenade®) was evaluated as the control antagonist, X. euvesicatoria Xp47 and Xe65  as pathogens, and X. campestris ATTC1395 as the negative control. Bacterial suspensions were prepared from 24 h growth cultures at 28 °C and 120 rpm in Luria Bertani (LB) liquid medium at pH 7.0. Bacterial density was adjusted to 1 ×108 CFU/mL, corresponding to 0.4 at 600 nm OD by UV-visible spectrophotometry  for Xanthomonas and Bacillus spp.  when the growth of at least one colony of the pathogen was detected in the reseeding, during the evaluation period (the “-” sign indicates no growth inhibition).lus spp. . BacteriXanthomonas by F-BC26, F-BC08, and F-BT24 strains. This test was established in a plant growth room with a photoperiod of 16 h light at 28 °C and 8 h dark at 18 °C and 80% to 90% RH. In this assay, we used 35-d-old jalapeño pepper plants variety M. Seedlings were cultivated in 10 cm diameter pots with sterile peat moss and irrigated every third day with the nutrient solution proposed by 8 CFU/mL) suspensions, whereas 10 mL of Serenade® (1 × 108 CFU/mL) or nutrient solution )We evaluated plant resistance to X. euvesicatoria Xp47 and Xe65 suspensions at 1 × 108 CFU/mL or 0.85% sterile saline solution (control). Before and after pathogens inoculation, plants were conditioned for two days in darkness, with an RH greater than 90%. Disease inhibition by inducing resistance was determined after 15 d of pathogens inoculation, using the . Plants without any inducing treatment were used as controls. After seven days, plants foliage (two points on two leaves per plant) was inoculated through infiltration with one milliliter Plastipak SFP syringes , using 10 µL of Where: 8 CFU/mL) plus 0.005% Tween 20, Serenade® (as a positive control at 1 × 108 CFU/mL), 10 mL of 50% Anglosan CL® (didecyldimethylammonium chloride (DDAC); American Pharma S.A. de C.V.), 10 mL of FitoFort® applied to the stem base (as resistance inducer), or 10 mL of sterile distilled water. After 48 h, approximately three milliliters of X. euvesicatoria Xp47 and Xe65 suspensions at 1 ×108 CFU/mL or sterile saline solution at 0.85% were sprayed on foliage as negative controls. Before and after pathogens inoculation, plants were conditioned for two days in darkness, with a RH higher than 90%. Xanthomonas spp. biocontrol by Bacillus spp. was evaluated after 15 d of pathogen inoculation and Bacillus positive antagonistic effect was recorded if disease symptoms were not observed in leaves, whereas Bacillus absence of antagonistic effect was recorded when disease symptoms on the foliage were observed. In addition, pathogen presence in leaf tissue was determined by taking samples, grinding them with a pestle, and taking microdilutions to inoculate on MacConkey Agar plates  as a positive control group, and X. euvesicatoria Xp47 and X. euvesicatoria Xe65 as the negative controls, and plants without formulations as controls. Resistance induction assays were performed five times, whereas foliar biocontrol experiments had four replicate determinations per treatment.Resistance induction in pepper against Bacillus- induced pepper plants growth with unformulated and formulated Bacillus spp. strains were analyzed by ANOVAs, and the Scott-Knott’s (α = 0.05) and the Tukey’s (α = 0.05) mean separation tests, respectively. Data from Xanthomonas spp. biocontrol assays were analyzed by ANOVAs and Tukey’s (α = 0.05) mean separation test. All analyzes were performed with the InfoStat software .Data from Bacillus spp. isolates. This step is recommended since these temperature range does not affect spore survival, but helps to eliminate other undesirable bacteria. After exposing samples at 90 °C by 10 min, mostly viable Bacillus spp. endospores will develop in the culture medium  and only one as B. thuringiensis. Isolates identification was confirmed with the 400 bp and 299 bp DNA fragments amplification of one isolate, using B. thuringiensis-specific primers, and 400 bp and 475 bp fragments amplification in isolates testing B. cereus-specific primers  improved after Bacillus spp. application (seedling emergence assay)  . After i control .The evaluated strains  increased the dry weight of roots, stems, and leaves by 46.1%, 45.4%, and 37.8%, respectively, as compared with the absolute control seedlings and commercial product. Regarding seedlings quality, expressed in terms of the Dickson Index, CBC08 and CBC26 strains improved plant quality by 60.7%, as compared with the absolute control seedlings . OverallP < 0.05) improved after applying formulated B. cereus and B. thuringiensis (Pepper seedlings grown under greenhouse conditions (seedlings emergence assay), significantly (ngiensis . FormulaP <  0.05) after applying formulated B. cereus and B. thuringiensis strains, as compared with fertilized plants (P < 0.05) improved leaf area, stem diameter, and leaf and stem dry weights by 82.0%, 20.6%, 62.9%, and 72.7%, respectively, as compared with the absolute control. F-BC26, F-BT24, and commercial product treatments, increased height and root dry weight by 48% and 141%, respectively, as compared with the absolute control. Furthermore, F-BC26 formulation and the commercial product improved the number of leaves by 41.1%, whereas only the F-BT24 formulation enhanced root length by 30.6%. Similarly, F-BC08 formulation improved chlorophyll “b” and carotenoid content by 157.7% and 33.0%, respectively, as compared with the commercial product. Regarding flowering stimulation, formulated F-BC26 strain significantly increased the number of flowers to 140.7%, as compared with fertilized plants (Potted pepper plants development under greenhouse conditions (vegetative stage) was significantly higher (d plants . The thrd plants .Bacillus isolates tested inhibited in vitro X. euvesicatoria Xp47 and Xe65 growth after 24 h of confrontation (X. campestris ATTC1395 was inhibited by Bacillus spp. after 24 h of confrontation (63% inhibition), but at 48 h, its growth was completely inhibited   (Formulated  < 0.05) . Similaribition) .Bacillus formulations or commercial products showed characteristic disease signs in treated plants but lower severity depending on the treatment. Necrotic spots with chlorotic edges presence characterized them, leaves with chlorosis or perforation areas with leaf blades, and necrotic spots with chlorosis . Overall, maximum pathogen population reduction by biocontrol agents was 8.6% . After liquid samples are prepared, they are “sprayed” inside of the tank where temperature is higher than 80 °C. Sample residence is of a few seconds, since it dries, and resulting micro-particles are vacuumed to a collector container. It has been proven that Bacillus spp. spores survive this spray drying process  and gibberellic acid. Previous studies have shown that B. cereus and B. thuringiensis improve pepper plants development by IAA production  iturin-like and fengicine of the surfactin family, have been widely documented as antimicrobial compounds produced by Bacillus spp.  and B. thuringiensis (CBT24) as PGPB, when applied during the emergence of pepper seedlings and crop development under greenhouse conditions. Formulations of F-BT24 and F-BC26 strains significantly promoted pepper seedlings growth and crop development. In vitro testing demonstrated that Bacillus isolates showed antagonistic potential against X. euvesicatoria. Under controlled condition experiment, F-BT24, F-BC26, and F-BC08 formulations efficiently controlled bacterial spot, similar to the commercial product (Serenade®). Based on these results, formulated B. cereus and B. thuringiensis may be used as biofertilizers and biocontrol agents against chili peppers bacterial spot. However, it is necessary to continue studying and evaluating their effects on yield and fruit quality.The present study demonstrated the potential of 10.7717/peerj.14633/supp-1Supplemental Information 1Click here for additional data file.


SHANK3 gene are causative for Phelan–McDermid syndrome (PMDS), a syndromic form of autism spectrum disorders (ASDs). We analyzed Shank3Δ11(−/−) mice and organoids from PMDS individuals to study effects on myelin. SHANK3 was found to be expressed in oligodendrocytes and Schwann cells, and MRI analysis of Shank3Δ11(−/−) mice revealed a reduced volume of the corpus callosum as seen in PMDS patients. Myelin proteins including myelin basic protein showed significant temporal and regional differences with lower levels in the CNS but increased amounts in the PNS of Shank3Δ11(−/−) animals. Node, as well as paranode, lengths were increased and ultrastructural analysis revealed region-specific alterations of the myelin sheaths. In PMDS hiPSC-derived cerebral organoids we observed an altered number and delayed maturation of myelinating cells. These findings provide evidence that, in addition to a synaptic deregulation, impairment of myelin might profoundly contribute to the clinical manifestation of SHANK3 deficiency.Mutations or deletions of the The online version contains supplementary material available at 10.1007/s00018-022-04400-4. Autism spectrum disorders (ASDs) are a group of neurodevelopmental conditions affecting approximately 1 in 68 people . The undSHANK3 leading to its haploinsufficiency [SHANK3 codes for a master scaffolding protein best known for its localization in the postsynaptic density (PSD) of excitatory synapses [One syndromic form of ASD is the Phelan–McDermid syndrome (PMDS), a rare neurodevelopmental disorder that accounts for 0.5% to 2.0% of ASD cases , 18. Theficiency , 21. SHAsynapses –24, whersynapses , 26. In synapses , 28.Shank3Δ11(−/−) mice using magnetic resonance imaging (MRI) analysis [Besides these synaptic changes, hippocampal and thalamic size have been reported to be changed in analysis . In addianalysis . Thus, Sanalysis , 31.Shank3Δ11(−/−) mice [Shank3Δ11(−/−) mice revealed a significantly reduced volume of corpus callosum, we further examined the myelin composition and the expression of myelin proteins in young and adult mice  in both the central (CNS) and peripheral nervous system (PNS). Lastly, our observations of alterations in myelin were confirmed by the analysis of hiPSC-derived cerebral organoids of PMDS patients. Based on these results, we propose that the symptoms observed in SHANK3-deficient patients are, at least in part, caused by altered myelin and do not exclusively arise from SHANK3 loss in neuronal synapses.This led us to speculate whether SHANK3 loss might change myelin. We used /−) mice  and cere/−) mice  from humThe human study was approved by the Ethics Committee of Ulm University . The experiments were conducted according to institutional and national guidelines and regulations. Prior to sampling, a written informed consent was provided by all the human donors and/or their legal guardians, as appropriate.Ethical approval for the animal experiments was obtained by the review board of the Land Baden-Württemberg, Permit Number o.103-12 and z.103 TschB:W. The experiments were performed in compliance with the guidelines for the welfare of experimental animals issued by the Federal Government of Germany, the National Institutes of Health, and the Max Planck Society.Mus musculus) were obtained from the animal research center of Ulm University. Knock-out Pro2 KO GVO (Shank3Δ11(−/−)) mice were housed as elucidated by [Shank3(+ / +)) or knock-out (Shank3Δ11(−/−)). For biochemical analysis 7-, 21- and 140-day-old male mice were used. For the MRI analysis four- and nine-week-old male and female animals were used.Wildtype C57BL/6JRj mice  and has been described previously . The anaSHANK3 point mutation. They have previously been characterized for pluripotency and have already been published [2 and 5% O2. Cells were scratched, washed and fed daily.All iPSC lines of this study have been reprogrammed from human keratinocytes. Seven different iPS cell lines were used for this study: three from healthy individuals, three from SHANK3 PMDS patients and one CRISPR/Cas9 engineered line carrying a ublished . hiPSC cCerebral organoids were generated following published protocols . They weShank3(+ / +) mouse pups. To obtain a mixed glial culture, the tissue was dissociated following published protocols [2 flasks coated with poly-l-lysine  diluted in borate buffer (Thermo). OPC medium  was changed every two to three days until a confluent monolayer has grown. Three days before purification the OPC medium was supplemented with bFGF (PeproTech Inc.) and PDGF-aa (R&D). After 15–20 days in culture, a monolayer mixture of astrocytes, microglia and OPCs was obtained. Flasks were shaken for 20 h at 250 rpm and 37 °C. Subsequently, the medium containing the detached OPCs was collected, and the cells were seeded at a density of 2.5 × 104 cells/cm2 onto poly-l-lysine-coated 12 mm wide glass coverslips (for immunocytochemistry experiments) and 12 × 104 cells/cm2 into poly-l-lysine-coated 6-well plates . On day 1 after purification, a full medium change was performed with proliferation medium , 100 µg/ml apo-transferrin, 100 µg/ml BSA, 40 µg/ml sodium selenite (Sigma), 60 ng/ml progesterone (Sigma), 16 µg/ml putrescine (Sigma), 5 µg/ml insulin (Merck), 10 ng/ml bFGF, 10 ng/ml PDGF-aa, 30 ng/ml T3, 40 ng/ml Thyroxine (Merck)). On day 2, proliferation medium was replaced by differentiation medium , FBS , 100 µg/ml apo-transferrin, 100 µg/ml BSA, 40 µg/ml sodium selenite (Sigma), 60 ng/ml progesterone (Sigma), 16 µg/ml putrescine (Sigma), 5 µg/ml insulin (Merck), 30 ng/ml T3, 40 ng/ml thyroxine (Merck)). After 6 days in differentiation medium, cells were ready for immunocytochemistry and lysates.Purified cultures of oligodendrocytes were prepared from total cortex of P0-P2 of wildtype rotocols  and the l-lysine-hydrobromide  coated plates at 37 °C. At 80% confluency, cells were stained or used for Western Blot lysates.Commercial primary mouse Schwann cells  isolated from 8-day-old C57BL/6 mice were used. They were passaged by incubating them with TrypLE (Gibco) for one minute at 37 °C. The flask was then softly knocked to detach the cells from the surface, 8 ml 1 × DPBS (Gibco) were added, and cells were transferred into a falcon. Cells were centrifuged at 1000 rpm for 5 min, re-suspended in Schwann cell medium  and were cultured on poly-The cells were washed with DPBS, fixed with 4% PFA (Merck)/0.1 M sucrose (Roth) in 1 × DPBS (Gibco) for 20 min at RT and washed three times for 5 min with 1 × DPBS. They were permeabilized with 0.2% Triton X-100 (Roche) diluted in 1 × DPBS for 10 min at RT. After blocking with 10% goat-serum (Millipore), 5% FBS (Gibco) in 1 × DPBS at RT for 4 h, the primary antibodies were incubated at 4 °C for 24 h. The following antibodies were used: anti-SHANK3 , anti-MALI-COs were washed with DPBS and fixed with 4% PFA (Merck)/0.1 M sucrose (Roth) in 1 × DPBS (Gibco) overnight at 4 °C. After the fixation, the individual sections were removed from the cell culture inserts, transferred to a 24-well tissue culture plate, and blocked with 5% FBS (Gibco) and 0.5% Triton X-100 (Roche) diluted in 1 × DPBS at RT for a minimum of 4 h. Sections were then immuno-labeled with corresponding antibodies at 4 °C for 48 h. The following primary antibodies were used: anti-MBP , anti-NeuN , anti-SHANK3 , anti-CFor immunostaining of the brain, spinal cord and sciatic nerve, mice were anesthetized by intraperitoneal injection of ketamine  and xylazine  in saline solution. Mice were trans-cardially perfused with 4% PFA  in DPBS. Collected tissue was post-fixed in 4% PFA overnight and cryoprotected with 30% sucrose in 1 × DPBS at 4 °C for 18 h. Tissues were afterward lodged in O.C.T. (Tissue-Tek), cut in transversal or longitudinal 16 µm (sciatic nerve), 20 µm  or 40 µm thick sections (brain) using a Leica CM1950 cryostat. Sciatic nerve sections were collected directly on microscopy slides, while free-floating brain and spinal cord slices were suspended first in 1 × DPBS, then in cryoprotectant . To use the sections, the cryoprotectant was removed and sections were washed three times with DPBS. The procedure was followed either by antigen retrieval with pre-warm 0.1 M citrate buffer (pH 6) at room temperature (RT) for 20 min  and Anti-Kv1.2 ) or were immediately blocked with 10% goat-serum (Millipore), 5% FBS (Gibco) and 0.1% Triton X-100 (Roche) diluted in 1 × DPBS/blocking solution at RT for 4 h. Sections were then immuno-labeled with corresponding antibodies at 4 °C for 48 h. The following primary antibodies were used: anti-MBP , anti-MBP , anti-NFH , anti-SHANK3 , anti-CShank3(+ / +) mice as described in the ‘immunohistochemistry’ chapter. After removal of cryoprotectant, sections were washed for three times with DPBS and incubated with primary antibodies diluted in blocking solution (3% BSA and 0.3% Triton X-100 in DPBS) twice, once pre-expansion and post-denaturation. Both times, the samples were incubated over the weekend at 4 °C with primary antibodies anti-SHANK3 (rb 1:250 homemade), anti-MBP , anti-NFH . Secondary antibody incubation was performed overnight at room temperature and a dilution of 1:200, but only post-denaturation. After the first primary antibody incubation, the TREx protocol was applied with minor adjustments to expand the samples [N,N′-methylenebisacrylamide and PBS−/− (1x) once the polymerization was initiated by the addition of TEMED (1.5 ppt) and APS (1.5 ppt). The polymerization process was slowed by 4-Hydroxy-TEMPO (22 ppm) to allow for an extended free-floating incubation time of 45 min in activated gelation solution, before mounting the slices into the gelation chamber and allow full polymerization for 1 h at 37 °C. Then, slices were recovered into digestion buffer  and incubated for 3 h at 80 °C in a Thermo-Block. After denaturation, the samples did undergo a second round of immunofluorescent staining as mentioned above. The stained gels were then incubated with DAPI  for 5 min at room temperature. Afterward the samples were washed 4–5 times in ddH2O and stored at 4 °C overnight to complete the expansion of the gel.Mouse brain sections undergoing expansion microscopy were obtained from  samples . BrieflyFor imaging, the region of interest was cut from the gel with a scalpel and mounted onto a #1.5H chambered cover glass (Cellvis). Images were acquired using a Leica TCS SPE II confocal microscope  equipped with a HC PL APO 63x/1,30 GLYC CORR CS2 glycerol-immersion objective. Images were deconvolved using Huygens Professional (SVI) version 21.04 and further processed using FIJI (ImageJ 1.53f51).Sciatic nerve cryosections were post-fixed with 4% PFA (Merck)/0.1 M sucrose (Roth) in 1 × DPBS (Gibco) for 15 min. Then they were permeabilized with 0.2% Triton X-100 (Roche) in 1 × DPBS for 1 h. Free-floating 40 µm thick brain as well as 20 µm spinal cord slices were permeabilized for 3 h. Afterward, the tissues were stained with FluoroMyelin Red (Invitrogen) diluted 1:300 in 1 × DPBS for 3 or 1 h, respectively. After washing 3 times with 1 × DPBS for 10 min each, the sections were mounted with ProLong Gold Antifade reagent with DAPI (Invitrogen). Overview images were acquired using a Leica DMi8 microscope. Detail images were acquired with a resolution of 1024 × 1024 pixels using the Leica TCS SPE II confocal microscope .Brain, cervical spinal cord, and sciatic nerve were mechanically homogenized in modified RIPA buffer (10 mM Tris–HCl pH 7.4 (AppliChem), 0.1% sodium dodecyl sulfate , 1% Triton X-100 (Roche), 1% sodium-deoxycholate (Merck), 5 mM EDTA (Sigma), protease/phosphatase inhibitor (Roche)) utilizing an electric tissue grinder and were lysed on ice for 15 min. The lysate was sonicated 10 times for 10 s and further incubated on ice for 30 min. Lysates were clarified by centrifuging at 13,000 rpm at 4 °C for 10 min. Protein concentration was determined via Bradford assay. Therefore, 20 µl of 150 mM NaCl (Merck), 2 µl of the vortexed sample and 200 µl Bradford solution , phosphoric acid , Serva Blue (Serva) solved in distilled water) were pipetted as duplicates into a 96-well plate (Sarstedt). The plate was placed into a microplate reader and the absorbance was measured at 595 nm. The measured values were used to calculate the protein concentration of the sample and were prepared in water and sodium dodecyl sulfate ACS reagent  according to the desired load. Samples were boiled at 95 °C for 5 min. Gel electrophoresis was performed at 90 V for about 15 min and then at 110 V for further 45 min. The protein was blotted onto a nitrocellulose membrane by a Trans-Blot® Turbo™ Transfer System (BioRad), the membranes were blocked with 5% skim milk powder (Sigma) in Tris-buffered saline with 0.1% TWEEN-20 (TBST). The membranes were incubated with primary antibodies at 4 °C overnight. The following primary antibodies were used: anti-SHANK3 , anti-MFollowing cervical dislocation, brain, spinal cord and sciatic nerve were dissected from the mice and fixed in pre-cooled 2% paraformaldehyde , 2.5% glutaraldehyde , 0.1 M Sorensen’s phosphate buffer, 1% saccharose at 4 °C for 24 h. The tissues were cut into small fragments and further fixed with 2.5% GA, 0.1 M Sorensen’s phosphate buffer, 1% saccharose at 4 °C for 24 h. Afterward, the tissues were post-fixed in 2% aqueous osmium tetroxide (Fluka), followed by stepwise dehydration of the samples with propanol. Thereafter, the samples were embedded in epoxy resin (Fluka). Semi-thin sections of 500 nm were cut and post-stained with 1% toluidine blue (Fluka) and 1% Na–borate (Sigma) in water. Resin blocks were trimmed, followed by cutting into ultra-thin 80 nm sections and placing them on copper grids 300mesh (Plano). Finally, the sections were contrasted with 0.3% lead citrate (Plano Agar). Images were taken using a JEOL 1400 Transmission Electron Microscope (TEM) and a Veleta camera (Olympus).For thickness measurements in cortex and corpus callosum in MBP stained slices, distances were measured from the mid-line of the brain as indicated in the picture.Shank3(+ / +) and three Shank3Δ11(−/−) animals or sections of one Shank3(+ / +) and Shank3Δ11(−/−) animal each.To ascertain FluoroMyelin, MBP, MPZ and SHANK3 intensity of brain, spinal cord, and the sciatic nerve, 5–6 confocal images per animal were analyzed measuring the mean gray value of the positive area per picture (sciatic nerve) or region of interest  using Fiji ImageJ . The maximum projection of all pictures was created in Fiji ImageJ. Within the NFH and MBP channel, a threshold, dependent on the intensity histogram and covering all the signal was set and a corresponding mask created. These masks of the first channel were overlapped to the SHANK3 channel, the second channel of interest, to determine the SHANK3 intensity only in the NFH-positive or MBP-positive area, respectively. Images of one experiment containing all experimental groups were always imaged together to ensure comparability. Since the absolute intensities varied between experiments, data were normalized to the mean of all data points obtained from the same experiment. One experiment included either sections of three To assess the complexity of the MBP staining in cortex, the number of intersections (intersection density (100 × 100px)) was analyzed using the ImageJ plugin “Diameter J” as described previously .Paranode, node and juxta-paranode analyses of transversal brain and longitudinal spinal cord sections were analyzed by acquiring six confocal images per animal. To evaluate paranode and node length the “straight line” tool was applied using Fiji ImageJ. Nine CASPR/Kv1.2 and node segments were analyzed per image. Each Kv1.2 fragment of a CASPR segment was counted and classified as adjacent or overlap.Paranode, node and juxta-paranode analyses of the sciatic nerve were conducted by taking 3–6 confocal pictures per animal. To measure the CASPR and the node length the “straight line” tool was applied using Fiji ImageJ. For P7: 30–180 CASPR/ Kv1.2 and 15–90 node segments per mouse, 4–86 CASPR/Kv1.2 structures and 2–43 node segments per slice; P21: 42–166 CASPR/Kv1.2 and 21–83 node segments per mouse, 20–46 CASPR/ Kv1.2 structures and 10–23 node segment per slice and for P140: 64–150 CASPR/Kv1.2 and 32–75 node segment per mouse, 10–38 CASPR/Kv1.2 structures and 5–19 node segment per slice were analyzed, respectively. Each Kv1.2 fragment of a CASPR segment was counted and classified as adjacent or overlap.MBP and NeuN positive cells in cerebral organoids were counted considering three regions of interest per image. Values were set relative to the total amount of DAPI nuclei per area.MBP , CC1 and CNP (slice organoids) intensity in hiPSC-derived cerebral organoids were analyzed by measuring the mean gray value of the positive area per region of interest using Fiji ImageJ. Within the CC1 channel (slice organoids) a threshold was applied, creating the respective mask. Thus, SHANK3 intensity was analyzed only in the CC1-positive area. Data were normalized to the mean of controls. Overview tile scan images were acquired on a Leica DMi8 microscope and high-resolution images were obtained using a Leica TCS SPE II confocal microscope .Electron microscopic images from brain, spinal cord and sciatic nerve were analyzed using the “free hand selections” tool in Fiji (ImageJ) to measure the area of an axon or the entire fiber. From these values the axon diameter and g ratio  were calculated in Microsoft Excel.For analysis of expansion microscopy, we analyzed transversal axonal section using Fiji image J. Three consecutive steps were collapsed as maximum intensity projection. The outer-most layer of the MBP signal was surrounded and selected as ROI to measure one complete fiber at a time. Within that ROI, the integrated intensity of the SHANK3 channel was measured. This value of SHANK3 content was set to 100%. Then, the area covered by the MBP signal was saved as a selection. This selection was put on the SHANK3 channel and the integrated density of SHANK3 was measured within this MBP mask. The SHANK3 content localizing within the MBP mask was calculated as percentage of SHANK3 in the complete fiber.The processed measurements were plotted with GraphPad Prism 8.t test (parametric data) or Mann–Whitney test (non-parametric data) were used to compare two groups. Categorized data with more than two classes were tested with Fisher’s exact test using R version 4.1.0 and rstatix package. Significance levels  were set to 0.05  with 95% confidence interval.Statistical analyses of data and graphs were performed using GraphPad Prism 8 Software and/or Microsoft Excel. Data display the mean ± Standard Error of Mean (SEM). Data were checked for normality using Shapiro–Wilk normality test. Two-tailed Student’s Shank3Δ11(−/−) mice, we performed structural MRI analysis and found a significant decrease in the volume of corpus callosum compared to Shank3(+ / +) mice  140-day-old (P140) mice . To specifically assess the myelin in the corpus callosum, brain tissue was stained with FluoroMyelin dye, and the staining intensity was found significantly reduced in Shank3Δ11(−/−) mice  mice but downregulated in P21 and P140 both in immunostaining  mice. Furthermore, SHANK3 protein expression was analyzed during development at P7 and P21 . As in P140 mice, a reduction of the isoforms a and c/d was observed in Shank3Δ11(−/−), whereas an increase of 300 kDa and e was seen at P7. The number of intersections in the MBP staining can be used to closely analyze MBP expression, since more details are incorporated in the analysis compared to signal intensity and area [Shank3Δ11(−/−) mice  mouse brains  mice, both morphologically with a decreased volume in corpus callosum and smaller striosomes in striatum, and on a molecular level with alterations in MBP expression.To assess potential white matter alterations in Shank3Δ11(−/−) mice  mice. CC1-positive cells expressed SHANK3 as well. Analysis of SHANK3 intensity in CC1-positive soma revealed residual but reduced SHANK3 expression in P140 Shank3Δ11(−/−) corpus callosum . The corpus callosum lacks dendrites and neuronal somas, suggesting that non-neuronal cells also express SHANK3. To exclude that the SHANK3 signal solely derived from axons, we performed expansion microscopy of P140 mouse brain sections. In the cortex  and in the corpus callosum, we found SHANK3 localizing in the axon, but also consistent SHANK3 expression in the MBP-positive myelin sheath surrounding the axon Fig. a. The pesum Fig. e, which Shank3Δ11(−/−) mice . In addition, the amount of Kv1.2-positive segments adjacent to CASPR was analyzed in P140 and a decrease was found in Shank3Δ11(−/−) in striatum and cortex . Thus, P21 and P140 Shank3Δ11(−/−) mice not only show a loss of SHANK3 and myelin proteins, but also changes in the node structure, suggesting possible alterations in signal propagation in these mice, since an increased node length has been associated with decreased axon conduction speed [To further investigate whether the SHANK3 and MBP loss in P21 and P140 mice might be associated with changes in the structure of the nodes of Ranvier, we immuno-stained for CASPR, a paranodal marker, and potassium channel 1.2 (Kv1.2), expressed in the juxta-paranodal zone  and Shank3Δ11(−/−) mice. FluoroMyelin intensity was decreased, among other areas, in the ventral white matter of Shank3Δ11(−/−) mice  .SHANK3 deficiency not only affects the brain but also all components of the voluntary motor system down to the muscle fiber  as well Shank3Δ11(−/−) mice  mice  mice  mice in the PNS. We also analyzed Myelin Protein Zero (MPZ), an important myelin protein in the PNS [Shank3Δ11(−/−) , we analyzed the sciatic nerve to study possible effects of SHANK3 deficiency. We evaluated the FluoroMyelin intensity in sciatic nerves of P7, P21 and P140 mice and observed an increase in  the PNS . We founShank3Δ11(−/−) mice , linking the myelin changes to SHANK3 deficiency. To confirm these findings on a cellular level, mouse primary Schwann cells were cultured. SHANK3 localized in these cells in immunostaining  mice in P21 and P140 mice . However, a significantly lower number of Kv1.2-positive structures was observed to localize with CASPR segments or being adjacent to them  mice [Shank3(+ / +) and Shank3Δ11(−/−) mice. The axon diameter remained unchanged , but the g-ratio was decreased  mice. In summary, in the PNS we observed increased MBP expression, but decreased MPZ in Shank3Δ11(−/−) mice. However, the thickness of the myelin was increased.SHANK3 expression was reduced in sciatic nerve protein lysate . We analyzed SHANK3 expression in sciatic nerve at P7, P21 and P140 in both the MBP-positive myelinating cells and the NFH-positive axonal compartments and found, again, a localization of SHANK3 in both compartments and reduced SHANK3 expression in /−) mice . FurtherSHANK3. In addition, we included a CRISPR/Cas9 engineered line (InsG) that, based on CTRL1, harbors an inserted guanine leading to a premature STOP codon in SHANK3  patient hiPSC lines that have a heterozygous deletion of ished in ) to ensuShank3Δ11(−/−) mice in myelinating cells, organoids were stained for CNP, and a reduced expression was found in PMDS  mice [Shank3Δ11(−/−) mice and hiPSC-derived organoids of PMDS patients and have, to our knowledge, performed the first study analyzing molecular changes in white matter under SHANK3 deficiency.This study is based on previous findings of MRI analysis in PMDS patients , 31 and /−) mice  with proShank3Δ11(−/−) mice could well resemble such accelerated myelination. The decreased volume of the corpus callosum we found in adult Shank3Δ11(−/−) mice has also been described in SHANK3-deficient macaques. These showed alterations in local and global connectivity patterns in MRI, indicative for circuit abnormalities [Myelination, the process of myelin formation, is crucial for the conduction velocity in fiber tracts in the nervous system and is necessary for the proper transmission of electrical signals in neuronal networks. Fiber tracts of the white matter link different brain regions enabling the control of cognition and behavior . Alteratmalities . In addimalities . These dIn this study, we showed the expression of SHANK3 in oligodendrocytes and Schwann cells and, therefore, we suggest a cell autonomous effect in those cells that eventually leads to the profound alterations in myelin we observed in SHANK3 deficiency. These observations indicate that the role of SHANK3 in neural tissue has been underestimated so far, since mainly the gray but not the white matter has been studied. The alterations of myelin under SHANK3 deficiency described in this study provides good evidence to further develop our understanding of SHANK3 in myelination. To date, PMDS has mainly been seen as a synaptopathy, but should eventually be considered as a combination of synaptopathy and myelinopathy. Of note, we did not observe any changes in axon diameter in any of the tissues analyzed. Nevertheless, it has been reported that axons are affected by SHANK3 loss. Neurite arborization was diminished in SHANK3-deficient primary hippocampal neurons , and alsThe putative role of SHANK3 in myelination is still unknown, however, myelin-associated changes have repeatedly been reported by the group of Han –57. PerfShank3Δ11(−/−) mice in most regions analyzed  is especially interesting. Speculating about possible reasons for the increased MBP expression at P7, one possibility would be that the lack of SHANK3 in its role as a scaffolding protein, both in precursors for neurons and oligodendrocytes, disturbs the development of the timed pattern of expression of downstream proteins and one of them might be MBP. At P7, a peak of oligodendrocyte proliferation is reached during development in mice [ASD is considered a neurodevelopmental disorder, meaning that neural alterations observed in patients might mainly derive from disturbances during development. Our organoid data revealed changes in the number of myelinating cells expressing MBP, CC1 and CNP already at an early age during neurodevelopment. In our hand, these organoids did not show myelination of axonal fibers yet, but the presence of oligodendrocyte progenitor cells (OPCs) has been shown before . In our  in mice , that go in mice . These eShank3Δ11(−/−) mice at P7. Since it is not known whether this increased myelination also occurs in SHANK3-deficient PMDS patients, it would be of interest to investigate brain and corpus callosum size in PMDS patients under the age of two. To date, MRI studies have been performed in small cohorts of patients with PMDS. Soorya et al. have revealed changes in corpus callosum in approximately 50% of the PMDS patients investigated that included thinning or hypoplasia, white matter changes, delayed myelination and generalized white matter atrophy [Shank3Δ11(−/−) mice might give a possible explanation for these myelin alterations in PMDS. Furthermore, it reveals that the common finding of atypical myelination in ASD has distinct and jointed traits in regard to the underlying etiology. However, longitudinal studies investigating white matter alterations in PMDS patients are needed to understand the dynamics of pathology progression.In other ASD models, MBP-associated changes have already been reported. A dynamic expression of MBP during neurodevelopment that is normalized in adult mice, has been reported in several ASD mouse models. P7 mice modeling Fragile X Syndrome demonstrated a reduced volume of the cerebellum together with an 80% reduction of MBP expression . However atrophy . Thinnin atrophy –64. The Shank3Δ11(−/−) mice by other myelin proteins, that have not been included in this study.Besides increased myelin and MBP at P7, we found decreased myelin levels in corpus callosum, cortex and spinal cord in adult animals. In spinal cord, the thickness of the myelin sheath was decreased on an ultrastructural level accordingly, while in corpus callosum such alterations were not visible. Alterations of MBP expression usually are reported to be accompanied by changes in myelin thickness and g-ratio, respectively , 66. MBPShank3Δ11(−/−) mice. Mature oligodendrocytes maintain the capacity to adjust paranode length and node elongation is a pathological phenomenon associated with myelin loss or paranodal changes (summarized in [Shank3Δ11(−/−) mice, that might also reflect in regression of PMDS patients. Therefore, the loss of MBP might directly impact on the lengths of node and paranode.While the increased MBP content we observed at P7 could explain the delayed achievement of developmental milestones in PMDS, that include motor function, cognitive abilities, and social achievements , 69, therized in ). These Shank3Δ11(−/−) mice, might be driven by the increased MBP expression. Since MPZ is expressed prior to MBP, an MPZ deficiency may be over-compensated by later synthesized myelin proteins such as MBP [In contrast to the CNS, we observed increased myelin and MBP expression together with decreased myelin protein zero (MPZ) expression in the PNS at all ages analyzed. In adult P140 mice, the thickness of the myelin sheath was increased. Myelination in the PNS is arranged fundamentally differently from the CNS, even though the resulting myelin and enwrapping of axons are ultra-structurally comparable. Schwann cells derive from the embryonic neural crest , and each as MBP  leading h as MBP ). This lWe found SHANK3 expression in both oligodendrocytes and Schwann cells. However, the expressed SHANK3 isoforms seem to be different, suggesting that the expression of distinct SHANK3 isoforms might affect the expression of MBP differentially, leading to a decrease in the CNS and an increase in the PNS. Ultimately, SHANK3 deficiency leads to loss of MBP in the CNS and to loss of MPZ in the PNS, both being important myelin proteins of the respective tissues. So far, we can only speculate how the loss of SHANK3 indeed might lead to deficits in myelination, differentiation and function of oligodendrocytes. Given the known scaffolding role of SHANK3 in neurons, SHANK3 might function similarly in myelinating cells and a loss of stability might contribute to the alterations we observed. SHANK3 might anchor receptor molecules that then get lost under SHANK3 deficiency. The resulting lack could then result in the observed changes in myelination and the expression of MBP. In respect of development, the interplay between neurons and myelinating cells is essential for the proper formation of connections. One could speculate that the loss of SHANK3 in neurons also impacts on the differentiation of oligodendrocytes and Schwann cells, demanding the analysis of the interaction of the two cell types in future experiments.Shank3Δ11(−/−) mice in the PNS might hinder the accurate signal transmission from the periphery to CNS. Furthermore, it might explain the general delay in development of patients with PMDS, assuming that signal integration of sensory information from the periphery is essential for proper formation of circuits in the central nervous system. MRI studies showed that both commissure and projection fibers are affected in PMDS [In respect of patients with PMDS, a variety of comorbidities can be attributed to those myelin-related changes we observed. For example, SHANK3-deficient patients manifest with a decreased perception of pain , 69. The in PMDS . The posSupplementary Figure 1. a MRI analysis of corpus callosum volume in 4-week-old Shank3(+/+) and Shank3Δ11(−/−) animals. Males: n=10 +/+, n=5 −/− animals. Females: n=7 +/+, n=4 −/− animals Mean±SEM. Student’s Unpaired t-test, total p=0.0075, right p=0.0153, left p=0.0048, total males p=0.1523, right males p=0.2914, left males p=0.0892, total females p=0.0151, right females p=0.0106, left females p=0.0212. b MRI analysis of corpus callosum volume in 9-week-old Shank3(+/+) and Shank3Δ11(−/−) animals. n=10 +/+, n=5 −/− animals. Mean±SEM. Student’s Unpaired t-test, total p=0.0009, right p=0.0020, left p=0.0006, total males p=0.0211, right males p=0.0333, left males p=0.0149, total females p=0.0132, right females p=0.0206, left females p=0.0119. c Total brain volume analysis in 4-week-old Shank3(+/+) and Shank3Δ11(−/−) animals. n=10 +/+, n=5 −/− animals. Mean±SEM. Student’s Unpaired t-test, total p=0.0156, males p=0.1843, females p=0.0046. Total brain volume analysis in 9-week-old Shank3(+/+) and Shank3Δ11(−/−) animals. n=10 +/+, n=5 −/− animals. Mean±SEM. Student’s Unpaired t-test, total p=0.2736, males p=0.5036, females p=0.2798. d MBP IHC and analysis of gray and white matter thickness of 140-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. Scale bar 500μm. n=3 animals. Mean±SEM. Student’s Unpaired t-test. Corpus callosum: distance was measured from the mid-line up to 1200μm. Mid-line: p=0.0226, 200μm p=0.0222, 400μm p=0.0033, 600μm p=0.0098, 800μm p=0.0272, 1000μm p=0.2496. 1200μm p=0.4404. Cortex: 200μm p=0.6818, 400μm p=0.5539, 600μm p=0.2489, 800μm p=0.0953, 1000μm p=0.3735, 1200μm p=0.2858. Supplementary Figure 2. a Western Blot analysis of SHANK3 and β-ACTIN in corpus callosum of 7-, 21- and 140-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. n=3 animals. Mean±SEM. Student’s Unpaired t-test. P7: 300kDa p=0.0628, c/d p=0.0011, e p<0.0001. Mann-Whitney test. P7: a p=0.1000 P21: 300kDa p=0.8707, a p=0.00734, c/d p=0.0009, e p=0.1764; P140: 300kDa p=0.0831, a p=0.0044, c/d p=0.0011, e p=0.0254. b FluoroMyelin intensity analysis in cortex, striatum of 140-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. n=4 animals. Mean±SEM. Student’s Unpaired t-test, cortex: p=0.0005, striatum: p=0.3498. c MBP IHC in corpus callosum, cortex and striatum of 7-, 21- and 140-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. Scale bar 30μm. d Western Blot analysis of MBP and β-ACTIN in cortex of 7-, 21- and 140-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. n=3 animals. Mean±SEM. Student’s Unpaired t-test. P7 p=0.0009, P21 p=0.6847. P140 p=0.0584. e Western Blot analysis of MBP and β-ACTIN in striatum of 7-, 21- and 140-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. n=3 animals. Mean±SEM. Student’s Unpaired t-test, P7 p<0.0001, P21 p=0.4945, P140 p=0.8008. Supplementary Figure 3. a SHANK3 and MBP IHC in corpus callosum of 140-day-old Shank3(+/+) animals. Scale bar 50μm. b SHANK3, MBP and NFH IHC in cortex of 140-day-old Shank3(+/+) animals using expansion microscopy. The percentage of SHANK3 in the MBP compartment relative to the total SHANK3 content per fiber is shown. Scale bar 10μm. n=4 animals. Mean±SEM. Student’s Unpaired t-test, p=0.0480. c ICC for O4 and DAPI in primary oligodendrocytes. d SHANK3 and CC1 IHC and analysis in corpus callosum of 7- and 21-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. Scale bar 15μm. n=3 animals. Mean±SEM. P7 Mann–Whitney test CC1+ p=0.100, P21 Student’s Unpaired t-test CC1+ p=0.0082. e TEM analysis of corpus callosum of 140-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. g-ratio of individual axons as a function of axon diameter. Shank3(+/+) R²=0.0257, Shank3Δ11(−/−) R²=0.02904, Slope: p<0.0001, Intercepts: Shank3(+/+) = 0.727, Shank3Δ11(−/−) = 0.7602. Axonal diameters were measured. n=4 animals. Mean±SEM. Student’s Unpaired t-test, p=0.2493. f CNP and DAPI IHC and analysis in corpus callosum, cortex and striatum of 21- and 140-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. Scale bar 30μm. n=3 animals. Mean±SEM. Corpus callosum P21 Student’s Unpaired t-test p=0.0581. P140 Mann–Whitney test p=0.100. Cortex P21 Student’s Unpaired t-test p=0.0467, P140 Student’s Unpaired t-test p=0.7333. Striatum P21 Student’s Unpaired t-test p=0.0153, P140 Mann–Whitney test p=0.1000. Supplementary Figure 4. a CASPR and Kv1.2 IHC and analysis in cortex of 21- and 140-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. Scale bar 2μm. n=3 animals. Mean±SEM. Student’s Unpaired t-test, P21 paranode p=0.0372, node p=0.4507. P140 paranode p=0.0252, node p=0.0107, adjacent p=0.0634. b CASPR and Kv1.2 IHC and analysis in striatum of 21- and 140-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. Scale bar 2μm. n=3 animals. Mean±SEM. Student’s Unpaired t-test, P21 paranode p=0.6024, node p=0.2235, P140 paranode p=0.0219, node p=0.0112, adjacent p=0.0179. c CASPR and Kv1.2 IHC and analysis in corpus callosum of 140-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. Scale bar 2μm. n=3 animals. Mean±SEM. Student’s Unpaired t-test. Adjacent p=0.9695. Supplementary Figure 5. a+b Western Blot analysis of SHANK3 and β-ACTIN in cervical spinal cord of 140-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. n=3 animals. Mean±SEM. Student’s Unpaired t-test, 300kDa p=0.0719, a p=0.0559, e p=0.0587, f p=0.0002. Mann–Whitney test, e1 p>0.9999. C Western Blot of SHANK3 and β-ACTIN of cortex of 140-day-old Shank3(+/+) animals. d+e Western Blot analysis of SHANK3 and β-ACTIN in sciatic nerve of 140-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. n=3 animals. Mean±SEM. Student’s Unpaired t-test, 300kDa p=0.0625, a p=0.0149, e p=0.0086, e1 p=0.1420, f p=0.0428. f SHANK3, MBP, NFH and DAPI IHC and analysis in cervical spinal cord of 7-, 21- and 140-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. Scale bar 10μm. n=3 animals. Mean±SEM. Student’s Unpaired t-test. P7 SHANK3 intensity p=0.0014, SHANK3/MBP p=0.0004, SHANK3/NFH p=0.0034. P21 SHANK3 intensity p=0.0113, SHANK3/MBP p=0.0607, SHANK3/NFH p=0.0050. P140 SHANK3/MBP p=0.0104, SHANK3/NFH p=0.0183. g TEM analysis of ventral cervical spinal cord of 140-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. n=4 animals. Mean±SEM. Axonal diameters were measured. Student’s Unpaired t-test, p=0.3572. Supplementary Figure 6. a SHANK3, MBP, NFH and DAPI IHC and analysis in sciatic nerve of 7-, 21- and 140-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. Scale bar 5µm. n=3 animals. Mean±SEM. P7 SHANK3 Mann–Whitney test p=0.0100. SHANK3/MBP Student’s Unpaired t-test p=0.0229, SHANK3/NFH Student’s Unpaired t-test p=0.2390. P21 SHANK3 Mann–Whitney test p=0.0100. SHANK3/MBP Mann–Whitney test p=0.4000, SHANK3/NFH Student’s Unpaired t-test p=0.2362. P140 SHANK3 Student’s Unpaired t-test p=0.0476. SHANK3/MBP Student’s Unpaired t-test p=0.0471, SHANK3/NFH Student’s Unpaired t-test p=0.3827. b CASPR IHC and analysis in sciatic nerve of 21- and 140-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. Scale bar 2µm. n=3 animals. Mean±SEM. Student’s Unpaired t-test. P21 paranode p=0.1410, node p=0.3180. P140 paranode p=0.3552, node p=0.2560. c TEM analysis of sciatic nerve of 140-day-old Shank3(+/+) and Shank3Δ11(−/−) animals. Axon diameter was measured. Scale bar 5μm. n=4 animals. Mean±SEM. Student’s Unpaired t-test, p=0.9020. The correlation between g-ratio and axonal diameter is showed. Shank3(+/+) R²=0.4007, Shank3Δ11(−/−) R²=0.3462, Slope: p=0.6381, Intercepts: p<0.0001. 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Streptomyces sp. T7, was isolated from soil by our lab. The endo-β-1,4-xylanase (xynST7) gene was found in the genome sequence of strain T7, which was cloned and expressed in Escherichia coli. XynST7 belonged to the glycoside hydrolase family 10, with a molecular mass of approximately 47 kDa. The optimum pH and temperature of XynST7 were pH 6.0 and 60 °C, respectively, and it showed wide pH and temperature adaptability and stability, retaining more than half of its enzyme activity between pH 5.0 and 11.0 below 80 °C. XynST7 showed only endo-β-1,4-xylanase activity without cellulase- or β-xylosidase activity, and it showed maximal hydrolysis for corncob xylan in all the test substrates. Then, XynST7 was used for the production of xylo-oligosaccharides (XOSs) by hydrolyzing xylan extracted from raw corncobs. The maximum yield of the XOS was 8.61 ± 0.13 mg/mL using 15 U/mL of XynST7 and 1.5% corncob xylan after 10 h of incubation at 60 °C. The resulting hydrolysate products mainly consisted of xylobiose and xylotriose. These data indicated that XynST7 might by a promising tool for various industrial applications.A xylanase-producing strain, identified as  F. FS. oliylanases b. These ylanases c, which E. coli BL21 (DE3). The recombinant protein was approximately 47 kDa, which was in agreement with the calculated molecular mass (46.6 kDa) of XynST7 , S. halstedii JM8 (Xys1L), S. fradiae var. k11 (SfXyn10), and A. terreus (BCC129) [The XynST7 exhibited the maximum activity at 60 °C, which was in agreement with certain xylanases from (BCC129) ,25,26,27(BCC129) . Thus, X2+, which is similar to other endo-β-1,4-xylanases from Streptomyces thermocarboxydus HY-15 and Cellulosimicrobium sp. HY-12 [+, Co2+, and Mg2+ (≤1 mM), while a drastic inhibition was observed in the presence of Fe3+, Fe2+, SDS, and EDTA in a concentration-dependent manner, in agreement with the data from Geobacillus thermodenitrificans A333, except for EDTA [+, Ca2+, and Ni2+, as reported in the xylanase from Streptomyces sp. 9 [The effects of metal ions and reagents on the activity of XynST7 are shown in p. HY-12 ,30. The for EDTA . Little es sp. 9 .S. thermocarboxydus HY-15 and Streptomyces sp. S38 [p-nitrophenyl-saccharide derivatives  was negligible, meaning no α-arabinofuranosidase, β-xylosidase, or α- or β-glucosidase activities were detected. These results clearly highlight the strict specificity of XynST7 toward polysaccharides containing β-1,4-xylopyranose units, which showed the monospecific characteristics of GH10 xylanases [The hydrolytic properties of XynST7 toward natural and artificial substrates are shown in  sp. S38 ,32. The ylanases . Thus, XKm, Vmax, and kcat values for various xylans, were evaluated. The XynST7 showed the highest Vmax value (approximately 678.14 U/mg) and lowest Km value (approximately 1.33 mg/mL) toward corncob xylan. Additionally, the respective Vmax and Km values of XynST7 were 596.54 U/mg and 2.78 mg/mL for birchwood xylan, 545.33 U/mg and 3.12 mg/mL for oat spelt xylan, and 495.23 U/mg and 3.54 mg/mL for beechwood xylan, respectively. The catalytic efficiency levels (kcat) of XynST7 for corncob xylan were higher by approximately 1.36-, 1.67-, and 2.08-fold than for birchwood, oat spelt, and beechwood xylan, respectively. Taken together, these findings indicate that XynST7 is a true endo-β-1,4-xylanase that exclusively cleaves the β-1,4-xylosidic linkages in different xylosic compounds.In this case, the kinetic parameters of XynST7, including Aspergillus foetidus MTCC 4898 [Xylan from the mild alkali extraction of raw corncobs was used for enzymatic hydrolysis to produce XOSs. As shown in TCC 4898 . AkpinarTCC 4898 . The prePenicillium funiculosum, Jonesia denitrificans, and Aspergillus fumigatus var. niveu, as well as over bacteria such as Thermobacillus composti, Paludibacter propionicigenes, and Streptomyces sp. B6 [XynST7 has a greater advantage for XOS production over other xylanases from fungi, such as s sp. B6 ,39,40,41Based on these results, XOS production was performed using 15 U/mL of XynST7 and 1.5% corncob xylan at 60 °C incubation for 24 h. The resulting XOSs  were analyzed via TLC. The most noteworthy was that only XOSs (xylobiose and xylotriose) were observed during the process of XOS production a, meaninE. coli DH5α and pMD 18-T vectors for gene cloning and E. coli BL21 (DE3) and pET-28a (+) for gene expression of endo-β-1,4-xylanase were preserved in our laboratory. Restriction endonucleases BamH I and Xho I, PrimeSTAR® HS DNA polymerase, and T4 DNA ligase were purchased from TaKaRa . The substrates pNP-α-d-arabinofuranoside (pNPA), pNP-β-d-xylopyranoside (pNPX), pNP-α-d-glucopyranoside (pNPGα), pNP-β-d-glucopyranoside (pNPGβ), carboxymethyl cellulose (CMC), and Avicel, as well as xylans from beechwood, birchwood, oat spelt, and corncob, were purchased from Sigma Chemical Company . All other chemicals used in the present study were of analytical grade.2PO4, 0.5% K2HPO4, and 0.5% MgSO4·7H2O. Subsequently, the microorganisms were gradually diluted with normal saline and screened using a primary screening medium . The xylan-producing colonies were screened through Congo Red staining and cultured on the fermentation medium . The hydrolysis activity of the fermentation supernatant was analyzed via thin-layer chromatography (TLC). The strain with the highest activity for xylan hydrolysis was identified based on the analysis of the 16S rRNA sequence, which was amplified using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′).Soil samples from the Colorful Desert  were collected and used for the enriched culture with liquid medium containing 1% beechwood xylan, 3% yeast extract, 0.5% KHhttps://services.healthtech.dtu.dk/service.php?SignalP-6.0, accessed on 3 January 2022). Protein homology searches were conducted using BLASTX . The multiple alignment was completed online using ClustalW  and ESPrist 3.0 . Conserved domains were analyzed using National Center for Biotechnology Information (NCBI) CD-Search . The classification of enzymes into GH families was carried out using InterPro . The theoretical molecular mass of the recombinant protein was predicted using the ExPASy ProtParam tool . The three-dimensional structure of xylanase from Streptomyces sp. T7 (XynST7) was predicted with a Swiss-Model server  using the xylanase from S. olivaceoviridis E-86 (PDB-ID: 1ISV) as the template and optimized based on the energy minimization.The presence of signal peptides was detected using the SignalP 6.0 server (BamH I restriction site) and reverse primer (5′-ATCTCGAGTCAGGTGCGGGTCCAGCGCTG-3′ with Xho I restriction site), and ligated into the pMD 18-T vector. The resulting pMD 18-T-xynST7 was transformed into E. coli DH5α and sequenced by Tsingke Biotechnology Co., Ltd. . The plasmid pMD 18-T-xynST7 was digested with BamHI and XhoI, and the resulting xynST7 was ligated to the pET-28a (+), which was transformed into E. coli BL21 (DE3). A single colony harboring the plasmid pET-28a-xynST7 was grown at 37 °C for 12 h in 3 mL of Luria–Bertani (LB) medium containing 50 µg/mL kanamycin. This culture was transferred to a fresh LB medium containing kanamycin according to the inoculation amount of 2% (v/v) and grown at 37 °C with shaking at 200 rpm. When the OD600nm value of the culture reached 0.6, xynST7 expression was induced by adding isopropyl-β-D-thiogalactopyranoside to a final concentration of 1 mM, then cultivation was continued for another 3 h at 37 °C. The xynST7 expression was confirmed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).The XynST7-encoding gene  was amplified using the forward primer  cells with xynST7 recombinant expression were disrupted by ultrasonication on ice . The supernatant was collected by centrifugation at 12,000× g for 20 min at 4 °C, and applied to a Ni Sepharose 6FF column following the manufacturer’s protocols . Protein fractions were eluted with 10 mM imidazole buffer first, then 100 mM imidazole buffer at a flow rate of 1ml/min. After dialysis for desalting, the collecting liquid was concentrated by ultrafiltration using a Millipore Ultrafiltration System with a molecular weight cutoff at 10 kDa . The purified protein was analyzed by SDS-PAGE. The molecular mass standards of protein were used in the range of 20–150 kDa .XynST7 was purified via a series of steps, including ultrasonication, Ni2PO4–Na2HPO4 buffer, pH 6.5) was incubated with 0.1 mL of enzyme at 60 °C for 30 min. The reaction was stopped using 0.6 mL of 3,5-dinitrosalicylic acid (DNS) reagent [pNPA, pNPX, pNPGα, and pNPGβ as substrates, respectively, and their reaction mixtures were incubated at 60 °C for 10 min, respectively. The absorbance of the released p-nitrophenol at 410 nm was determined. Xylanase activity was assayed using beechwood xylan as a substrate. Here, 0.3 mL of 1% xylan in 0.1 M PBS buffer (NaH reagent . The via3COONa-CH3COOH buffer (pH 4.0–6.0), Na2HPO4-NaH2PO4 buffer (pH 6.0–8.0), and Gly-NaOH buffer (pH 8.0–11.0)) at 60 °C for 30 min. Under stable pH conditions, the enzyme was pre-incubated at various pH values (pH4.0–11.0) at 4 °C for 24 h. For the optimum temperature, the enzyme activity was measured at various temperatures (20–80 °C) and pH 6.5 for 30 min. Under stable temperature conditions, the enzyme was pre-incubated at various temperatures (20–80 °C) with 5–60 min of incubation at pH 6.5. The effects of different metal ions and additives on enzyme activity were determined by incubating the enzyme with solution containing Na+, K+, Ca2+, Fe2+, Mg2+, Mn2+, Co2+, Ni2+, Fe3+, EDTA, and SDS for 24 h at 4 °C. Kinetic parameters were determined by performing enzymatic reactions at 60 °C, with various xylans (0.8–4 mg/mL) in 0.1 M NaH2PO4–Na2HPO4 buffer (pH 6.5) as the substrate. The catalytic constant (kcat) and catalytic efficiency (kcat/Km) were calculated using Km and Vmax values determined from the Lineweaver–Burk plot.For optimum pH, the enzyme activity was measured at various pH values (pH4.0 –11.0) using CHg for 20 min. The resulting supernatant was acidified to pH 5.0 with HCl, which was used as a substrate for enzymatic hydrolysis. The effects of various physico-chemical parameters, including the reaction time , enzyme dosage , substrate concentration , and incubation temperature , on the yield of XOS were successively investigated in order to maximize XOS production. Finally, XOS production was carried out by hydrolyzing corncob xylan under the optimum conditions. The resulting hydrolysates of corncob xylan were analyzed via thin-layer chromatography (TLC) on a silica gel 60 plate . The plate was developed twice using n-butyl alcohol/ethanol/water  and sprayed with a mixture of aniline diphenylamine and phosphoric acid, followed by heating at 90 °C for 10 min to detect sugar spots. The amounts of reducing sugars were quantified using the DNS method [Corncob xylan was prepared using a mild alkali extraction method, as previously described . The rawS method . To gather more detailed information of XOSs, HPLC analysis was performed on an HPLC system  equipped with a differential refractive index detector  and a CarboPAC analytical column , which was maintained at 60 °C using deionized water as the mobile phase at a flow rate of 0.2 mL/min. A mixture of xylobiose and xylotriose was used as the standard. xynST7) from Streptomyces sp. T7 was identified through genome sequence alignment and enzyme assay. XynST7 as a member of the glycoside hydrolase family 10 exhibited good pH adaptability and thermostability, while XynST7 had no cellulase activity, and it was able to efficiently hydrolyze xylans extracted from raw corncobs to produce xylo-oligosaccharides (XOS) without xylose, mainly being xylobiose and xylotriose. Therefore, XynST7 is of great value for further research and application developments.Xylanases are ubiquitous and diverse in the natural environment. Some of them have been used to decompose lignocellulosic materials in industry. Recently, cellulase-free xylanases have received great attention due to their environmental friendliness. Actinomycetes are particularly interesting producers of xylanases from an industrial point of view. In this paper, an endo-β-1,4-xylanase gene (


All of them showed two- to eightfold higher activity (minimum inhibitory concentration (MIC) 0.5–4 µg/mL) against the resistant strain compared with the reference drug. Compounds 5 and 7, which contained the most basic substituents—pyrrolidine and piperidine—in their structure, strongly inhibited the growth of the standard strain (MIC 2 µg/mL). Furthermore, the same derivatives exhibited activity comparable to that of the reference drugs against some types of Gram-positive bacteria (MIC 0.49 µg/mL) and showed no cytotoxicity (IC50 > 50 µg/mL) in HaCaT cells. The zwitterionic structure of each compound was determined using X-ray crystallography. Absorption, distribution, metabolism, and excretion analyses showed that all compounds are good drug candidates. Thus, compounds 5 and 7 were identified as leading structures for further research on antituberculosis drugs with extended effects.In this study, six new 2,6-disubstituted thiosemicarbazone derivatives of pyridine were synthesized ( Mycobacterium tuberculosis undec-7-ene (DBU) was added. The mixture was refluxed for 1–12 h. After the evaporation of the solvent, ice was added. The precipitated products were filtered, dried, and recrystallized from a suitable solvent or purified by column chromatography.1 was obtained as white crystals : m.p. 73–75 ℃ (methanol); IR (KBr): 3097, 3075 (υ CAr-H), 2959, 2865 (υ C-H), 2230 (υ C≡N), 1616, 1595 (υ C=N), 1499, 1458 (υ C=C), 1247, 1225, 1207, 1185 (δ C-H), 793 (γ C-H) cm−1; 1H NMR : δ 1.92–1.95 , 4.58–3.84 , 6.75 , 7.08 , 7.62  ppm; 13C NMR : δ 25.34 (2C), 46.85 (2C), 112.05, 116.69, 118.73, 130.93, 138.17, 157.15 ppm; Anal. Calcd for C10H11N3 (173.21): C, 69.34; H, 6.40; N, 24.26; Found: C, 69.38; H, 6.17; N, 24.52.Starting from 6-chloropicolinonitrile (5.5 g) and pyrrolidine (4 mL), compound 2 was obtained as slightly yellowish liquid : m.p.—(AcOEt:CHCl3 1:5); IR (KBr): 3019 (υ CAr-H), 2939, 2856 (υ C-H), 2234 (υ C≡N), 1595 (υ C=N), 1487, 1446 (υ C=C), 1254, 1215 (δ C-H), 771 (γ C-H) cm−1; 1H NMR : δ 1.51–1.54 , 1.0–1.61 , 2.50–2.51 , 7.11 , 7.15 , 7.64  ppm; 13C NMR : δ 24.54, 25.36 (2C), 45.60 (2C), 112.19, 117.41, 118.56, 130.75, 138.86, 159.04 ppm. The results of the elemental analyses are consistent with a description in the literature isopropanol–DMF–water (1:1:1 v/v) for 4. Diffraction measurements were carried out on an XtaLAB Synergy diffractometer, Dualflex, , with a Pilatus 300 K detector at low temperature (100 K) and with CuKα (1.54184 A) radiation. Diffraction data were processed using CrysAlis PRO . Crystal structure solution and refinement were carried out using SHELX [Single crystals of compounds suitable for X-ray diffraction were obtained by slow evaporation of the solvents at room temperature, from methanol–DMF .In in olex2  reduces The pharmacokinetic properties, drug-likeness, and absorption of the synthesized compounds were analyzed. An ADME analysis was carried out using the SwissADME service (Swiss Institute of Bioinformatics 2021  and BOIL1–3 (yield 91–92%). Subsequently, the nitrile groups were converted into methyl imidate groups. These reactions were carried out at reflux for 4 h in the presence of methanol and a catalytic amount of DBU. To obtain final products 4–9 (yield 21–86%), each of the methyliminoesters, which were in the solution form prepared in situ, was condensed with morpholine-4-carbothiohydrazide and pyrrolidine-1-carbothiohydrazide by heating at boiling temperature for 0.5–2.5 h. Carbothiohydrazides were obtained in the reaction between methyl hydrazinecarbodithioate and cyclic amines in water or alcohol, following the modified method described by Klayman et al. [1H NMR, and 13C NMR spectra, elemental analysis. The results of the spectral analysis were in accordance with the assigned structures. The 1H NMR and 13C NMR spectra of all compounds are provided in The synthesis route used in this study is outlined in n et al. . All theM. tuberculosis strains H37Rv and Spec. 210 is presented in 5 and 7 showed the highest ability to inhibit the growth of bacterial cells. The structure of these compounds contained pyrrolidine and piperidine, respectively, located at the C6 position of the pyridine ring, and pyrrolidine in the thiosemicarbazone chain. Compared with the MIC values of INH (0.125 and 8 µg/mL), compounds 5 and 7 showed values that were 16 times higher (MIC 2 µg/mL) for the standard strain; however, more importantly, compound 7 showed a values that was 16 times lower (MIC 0.5 µg/mL), and compound 5 showed an value that was 8 times (MIC 1 µg/mL) for the resistant strain. The presence of a phenoxy moiety in 8 and 9 (with lower basicity and a higher molecular weight than piperidine and pyrrolidine) at the 6-position on the pyridine ring and the presence of morpholine in 4, 6, and 8 (with lower lipophilicity and higher electron density than pyrrolidine) on the side chain decreased the antituberculosis potency for both the standard strain (MIC 8–16 µg/mL) and the resistant strain (MIC 1–4 µg/mL). These derivatives showed activity that was two- to eightfold higher against the resistant strain as compared with the reference drug INH. Interestingly, for all compounds, lower inhibitory titers were observed for the resistant strain compared with that of the standard strain.In vitro tuberculostatic activity against M. tuberculosis H37Rv strain is used as a reference strain in various microbiological tests, including drug resistance tests, and in the search for new antimycobacterial drugs. Whole-genome sequencing of the reference strain and clinical strains revealed considerable genetic diversity between M. tuberculosis clinical isolates. The absence of some genes has also been proven in the genome of M. tuberculosis H37Rv in comparison with the genomes of clinical strains, e.g., genes encoding adenylate cyclase (MT1360), glycosyl transferase (MT1800), oxidoreductase (MT1801), and others. This genetic diversity observed in studies comparing the H37Rv genome with the genomes of clinical isolates contributes to the differences in pathogenicity and virulence of the strains and can explain the phenotypic variation of the strains. In addition, this fact may explain the higher MIC of the reference strain compared with the clinical strains [The  strains ,34,35,364–7 and 9 showed higher bacteriostatic activity against all types of Gram-positive bacteria—except for B. cereus ATCC 10,876 for 5—than against tuberculostatic activity. For example, for compound 7, the MIC value for all Staphylococcus strains was 0.49 μg/mL, whereas for the M. tuberculosis standard strain, it was 2 μg/mL. Moreover, their growth inhibition potential was equal to or higher than those of the reference drugs CIP and VAN, respectively. Compound 8 showed moderate or weak (MIC 31.3–250 μg/mL) to no activity (MIC > 1000 μg/mL) depending on the strain. None of the tested derivatives showed inhibitory effects on the growth of Gram-negative bacteria strains. A similar relationship was observed for fungi, except for C. albicans ATCC 102,231 for 4, 5, and 7, and C. parapsilosis ATCC 22,019 for 7, with good (MIC 7.8–15.6 μg/mL) or moderate activity (MIC 250 μg/mL).In vitro antimicrobial activity is presented in 4, 5, and 7 showed no cytotoxicity against noncancer cells. Compounds 6 and 8 inhibited cell metabolic activity while maintaining cell viability, whereas compound 9 showed the same while losing cell viability. The selectivity index (SI) was calculated by comparing IC50 values and determined using the sulforhodamine B method, for noncancerous epithelial HaCaT cells against MIC values for M. tuberculosis standard strains. The nontoxicity of compounds 4–8 was indicated by SI values > 1.0. For compounds 5 and 7, which showed the highest tuberculostatic activity, the MIC value was more than 25 times lower, as compared with the concentration causing a cytotoxic effect against noncancer cells.Cytotoxic activity in HaCaT cell lines determined using the two methods is outlined in 4, 6–8, showed static disorder. For trigonal structures, namely compounds 4 and 6, the disorder was so significant that the SQUEEZE procedure had to be applied. Three structures, namely 4, 6, and 8, were solvates, but the positions of the water molecules were well-defined only in compound 8. Strong hydrogen bonds occurring in the studied compounds are presented in 5, 7, and 9 showed the same hydrogen bond system. A chain hydrogen bond of the N–H···S type was formed (9). A chain bifurcated hydrogen bond of the N–H···S···H–N type was formed in compound 6  show increased antibacterial activity. All these compounds exhibit the same hydrogen bonding systems. Compound 9 has a lower level of antituberculosis activity; this may be due to the introduced large phenoxy group. Compounds 5 and 7 can be identified as leading structures which additionally showed an effect on the standard M. tuberculosis strain and on other microorganisms and were nontoxic on nontumor cells.The synthesized compounds