PPP1R21 were linked to a neurodevelopmental disorder with hypotonia, facial dysmorphism, and brain abnormalities (NEDHFBA) with pediatric onset. Functional studies unraveled impaired vesicular transport as being part of PPP1R21-related pathomechanism. To decipher further the pathophysiological processes leading to the clinical manifestation of NEDHFBA, we investigated the proteomic signature of fibroblasts derived from the first NEDHFBA patient harboring a splice-site mutation in PPP1R21 and presenting with a milder phenotype. Proteomic findings and further functional studies demonstrate a profound activation of the ubiquitin–proteasome system with presence of protein aggregates and impact on cellular fitness and moreover suggest a cross-link between activation of the proteolytic system and cytoskeletal architecture (including filopodia) as exemplified on paradigmatic proteins including actin, thus extending the pathophysiological spectrum of the disease. In addition, the proteomic signature of PPP1R21-mutant fibroblasts displayed a dysregulation of a variety of proteins of neurological relevance. This includes increase proteins which might act toward antagonization of cellular stress burden in terms of pro-survival, a molecular finding which might accord with the presentation of a milder phenotype of our NEDHFBA patient.PPP1R21 acts as a co-factor for protein phosphatase 1 (PP1), an important serine/threonine phosphatase known to be essential for cell division, control of glycogen metabolism, protein synthesis, and muscle contractility. Bi-allelic pathogenic variants in The online version contains supplementary material available at 10.1007/s12035-023-03219-9. PPP1R21 gene, but until today, its exact molecular effect on PP1 remains elusive. Considering the crucial role of PP1 and its regulatory units, PPP1R21 is expected to also have an important effect in various cellular functions. Recent investigations describe a novel neurodevelopmental syndrome caused by bi-allelic PPP1R21 loss-of-function variants and imply a role for PPP1R21 within the endosomal sorting process or endosome maturation pathway [PPP1R21 gene have been linked to the neurological phenotype [PPP1R21 cases shows a broad spectrum of abnormalities. The majority of patients have a developmental delay/intellectual disability, coarse facial features with low set ears, muscle hypotonia, brain anomalies , feeding problems, ocular signs (strabismus/nystagmus), cardiac defects, and respiratory findings [The protein phosphatase 1 (PP1) is a serine/threonine phosphatase that is known to be essential for cell division , protein pathway . So far,henotype –8. The pfindings .PPP1R21 disease, we utilized proteomics. This approach was prompted by the fact that proteomics was demonstrated to be a suitable technique to identify processes taking place in neurological diseases [However, the underlying pathomechanism is still not fully understood. To address this lack of knowledge and to unravel pathophysiological processes related to the etiology of the diseases  and thushttp://gnomad.broadinstitute.org).DNA samples from the proband and his parents were subjected to whole exome sequencing. Libraries were created with an Illumina exome capture (38 Mb target) kit and sequenced with a mean target coverage of 80x . Data analysis was carried out in two stages, first searching for pathogenic/likely pathogenic variants in genes known to be associated with the clinical presentation (in silico panel). If no variants were identified, and given the known consanguinity in the family, the full exome was interrogated by applying a stringent criterion searching for homozygous highly damaging variants  absent in the control population  buffer, 1% SDS with cOmplete ULTRA protease inhibitor (Roche), and a sonication step with an ultrasound probe , followed by centrifugation at 4 °C and 20,000 g for 15 min. To precipitate the proteins, 3 ml of ice-cold acetone was added and incubated overnight at − 20 °C. After centrifugation at 4 °C for 20 min at 20,000 g, the supernatant was discarded, and the precipitated proteins were dried under a flow hood for a few minutes. Then, the dried protein pellet was dissolved by adding 8 M urea and mixing for one hour. After complete solubilization, the samples were diluted to 2 M urea using 10 mM ABC  buffer. To determine the protein concentration, a BCA (Pierce BCA protein assay kit) was performed according to the manufacturer's instructions. Reduction and carbamidomethylation of the samples were performed with 10 mM TCEP (tris-(2-carboxyethyl)-phosphine) for an incubation time of 30 min at 37 °C and with 15 mM IAA (iodoacetamide) for an incubation time of 30 min at RT, respectively. Proteins were hydrolyzed in solution with a trypsin to protein ratio of 1:100. Tryptic digestion was performed overnight at 37 °C and stopped the next day by addition of 99% formic acid (FA). Samples were desalted by solid phase extraction using C18 filter cartridges (Waters) by washing with 0.1% trifluoroacetic acid (TFA). This was followed by elution with 80% acetonitrile (ACN). The purified samples were dried using a vacuum concentrator. The concentration was adjusted to 1 µg/µl with 0.1% TFA.All proteolytic digests were checked for complete hydrolysation using a monolithic column separation  on an inert Ultimate 3000 HPLC (Dionex) by direct injection of 1 μg sample. A binary gradient  of 5–12% B in 5 min and then of 12–50% B in 15 min was applied at a flow rate of 2.2 μl/min and at 60 °C. UV traces were recorded at 214 nm .Mass spectrometric measurements were performed in data-independent acquisition (DIA) mode. For this purpose, each sample to be analyzed was mixed with an appropriate amount of iRT standard peptides (Biognosys). A total of 1 µg of each sample was used for analysis. The final hydrolysate was loaded onto an Ultimate 3000 Rapid Separation Liquid Chromatography (RSLC) nanosystem with a ProFlow flow control in line with a Fusion Lumos Tribrid mass spectrometer (both from Thermo Scientific). After initial loading, peptides were pre-concentrated on a trapping column  using 0.1% TFA at a flow rate of 10 µl/min. Subsequent sample separation was performed on a reversed-phase column  using a binary gradient: 3% solvent B (84% ACN with 0.1% TFA) for 10 min, a linear increase in solvent B to 35% for 120 min, and a linear increase in solvent B to 95% for 10 min followed by a linear reduction in solvent B to 3% for 5 min. Full MS scans were acquired from 300 to 1100 m/z at a resolution of 60,000 (Orbitrap) using the polysiloxane ion at 445.12002 m/z as the lock mass. The automatic gain control (AGC) was set to 5E5 and the maximum injection time to 20 ms. The full MS scans were followed by 30 DIA windows acquired at a resolution of 30,000 (Orbitrap) with AGC set to 1E6, a maximum injection time of 60 ms, and a normalized collision energy of 32 (HCD).www.uniprot.org), containing 20,374 entries, were selected as the proteome background. Processing settings were as follows: enzyme was trypsin, minimum and maximum peptide length were set to 7 and 52, respectively, and missed cleavages were set to 2. Carbamidomethyl for cysteine was set as a fixed modification. Acetyl (protein N-term) and oxidation of methionine were set as variable modifications. All settings related to library generation including tolerances, identification, filters, iRT calibration, and workflow were left at factory defaults. For relative quantification, the Top N max 3 option was chosen, meaning that for each protein the average of the 3 peptides identified with the highest intensity was taken to calculate the quantitative value for that protein. The analyzed data were matched against an in-house spectral library [The acquired data were imported into Spectronaut software (Biognosys). The human proteome data from UniProt  was carried out: cell pellets of PPP1R21-mutant and two controls fibroblasts were fixed with 2.9% glutaraldehyde/0.4 M phosphate buffered saline (PBS) and were processed with a Leica EM TP tissue processor with 1%-osmium-tetroxide and embedded in resin. For electron microscopy, ultrathin sections were contrasted with 3% lead citrate trihydrate with a Leica EM AC20 (Ultrastain kit II) and were examined using a Zeiss EM 109 transmission electron microscope equipped with a Slowscan-2 K-CCD-digital camera (2 K-wide-angle Sharp:eye).Myelin figures (MF) were analyzed at ultrastructural images (magnification × 4.400 and 12.000). MF were counted in all fibroblasts in which the nucleus was gated.PPP1R21-patient and two controls  utilizing the Proteasome Activity Assay Kit (Abcam ab107921) according to the manufacturer's instructions. To block proteasomal activity, cells were treated with 10 µM MG132 for 16 h. Cellular viability was monitored by measuring the metabolic activity of fibroblasts making use of an MTT-assay . The MTT assay way carried out as described previously [The activity of the proteasome was measured in cultured fibroblasts derived from the eviously .Immunoblot studies on whole proteins extracts of the PPP1R21-patient and two pooled controls were carried out as described previously  utilizinATF4 Hs00909569_g1, ATP6 Hs00232586_m1, BAG3 HS00188713_m1, BECN1 Hs00186838_m1, CALRHs00376764_m1, CALU Hs04992290_s1, CANX Hs01558409_m1, EDEM1 Hs00976004_m1, EIF2A Hs00230684_m1, EIF2AK3/PERK Hs00984003_m1, ERN1/IRE1 Hs01086607_m1, GRP170/HYOU1 Hs00197328_m1, HSPA1B Hs01040501_sH, HSPA5/GRP78/BIP Hs00607129_gH, HSPA8 Hs03044880_gH, HSPB8 Hs00205056_m1, HSP90B1/GRP94 Hs00427665_g1, LAMP1 Hs00931461_m1, LC3 / MAP1 Hs01076567_g1, LMP7/PSMB8 Hs00544760_g1, PDIA2 Hs00429010_m1, PDIA3 Hs00607126_m1, PGK1 Hs99999906_m1 (reference gene), PSME1 Hs00389210_g1, PSME2 Hs01923165_u1, SEC63 Hs00273093_m1, and XBP1 Hs00231936_m1.Total RNA was extracted from fibroblasts of the patient, as well as controls, using a Trizol-chloroform method and reversely transcribed into cDNA using the High-Capacity cDNA Archive Kit . For qPCR reactions, 10 ng of cDNA was used for analysis on the QuantStudio 6 Flex System  with the following running conditions: 95 °C 0:20, 95 °C 0:01, 60 °C 0:20, and 45 cycles . All targeted transcripts were run as triplicates. The TaqMan® Gene Exp Assay (Life Technologies/ ThermoFisher) is listed as follows: p value < 0.05 was considered significant and calculated with GraphPad Prism 9.2.0 .Gene expression of patient fibroblast was determined three times and illustrated by the fold change compared to the mean level of control fibroblasts. A High resolution scans of human fibroblasts stained with FITC-phalloidin were performed using a Leica TCS SP8 confocal laser scanning microscope with acousto-optic tunable filters, an acousto-optical beam splitter, internal hybrid detectors (HyD SP), and a LMT200 high precision scanning stage. Imaging of coverslip-embedded samples was performed via a Leica HC PL APO 63x/1.20 W CORR objective in combination with variable digital zoom factors according to fibroblast size. Phalloidin-AlexaFluor488 labeled actin fibers were excited with an argon laser at 488 nm and detected with an internal HyD at 500–550 nm. In a second sequence, DAPI was excited with a 405 nm diode-pumped solid-state laser and detected by an internal HyD at 450–500 nm. Generated images were deconvoluted with Huygens Professional (SVI) and reconstructed with Imaris software (Bitplane). To generate the circular color map and the coherency orientation index (COI), the confocal scanned z-stacks were loaded into ImageJ and converted into maximum intensity projections (MIP) using the Z Project tool. Single cells were identified by manual region of interest (ROI) definition and optically separated by clearing the area outside the ROI. Afterwards, the isolated cells were horizontally aligned, the color maps were generated, and the COI of the respective cells was determined with the help of the OrientationJ plugin. Statistical analysis of the COI values was performed with GraphPad Prism 9.The filopodia of human fibroblasts were manually isolated optically from the rest of the actin-fiber skeleton of the cell body based on their morphological characteristics and quantified accordingly. The length as well as the branching of filopodia was quantified using the ImageJ plugin "Analyze Skeleton (2D/3D)" .z-scores and percentiles: [The patient, a 6-year-old boy, is the first child of healthy consanguineous parents of Turkish origin. Pregnancy was uneventful, mother being 24 years old at conception. Birth was an uncomplicated with spontaneous delivery at 38 + 1 weeks of gestation . Birth measures were as following: weight = 3640 g , length = 53 cm , and head circumference = 33,5 cm  (entiles: ).At the age of 2 h, the patient was admitted to the neonatal care unit due to a cardiopulmonary adaption disorder. He needed respiratory support via high flow nasal cannula (HFNC) and intravenous fluids for two days. Directly postnatally, an intermittent strabismus of both eyes, a slight mandibular retrognathia, a slight thoracic instability, and low-set ears were noticed. Muscle tone was truncal hypertonic and peripheral hypotonic. Magnetic resonance imaging (MRI) of the head showed no malformations , electroencephalography (EEG), and echocardiography showed normal results. Visually evoked potentials were abnormal but ophthalmologic examination showed normal results.At 7 months of age, the parents reported good thriving. In the clinical examination, the child showed a generalized hypotonia. In the traction test, the head was taken along only briefly, in prone position also only brief head control was possible. No weight assumption in held stance. Social smile, vocalization, and normal interaction with his mother were noticed. Strabismus convergens alternans was still present.At the age of one year, the parents reported a developmental delay. He had not yet learned to crawl, sitting without support was not possible. He could roll from prone to supine. The MRI follow-up showed a moderate periventricular leukomalacia (PVL).2  , the face broad. Pupillary reaction to light was regular on both sides, he followed a light source. During sleep, the eyelids were half open. The bridge of the nose was narrow, as were the lips; the philtrum was long. The ears were large and low set, slightly asymmetrical. The muscle tone was markedly hypotonic. No raising of arms, no grasping was shown. He was able to turn from supine to prone position. Build was strong, trunk to limb ratio proportionate. Skin was fair, no pigment abnormalities present, the patient had dark blond hair. The hands showed a single transverse palmar crease. Chest was flat, heart and lungs auscultation were unremarkable, no liver or spleen enlargement existing. Genitals were male, prepubertal, right testis was descended, left not palpable (cryptorchidism). Body weight 20 kg , body height 104 cm , BMI 18.5 kg/mcentile: ).Based on the lack of the full clinical presentation of NEDHFBA known from the literature , especially regarding cardiac abnormalities, scoliosis, and CNS anomalies, we postulate the presence of a milder phenotype in our patient.http://gnomad.broadinstitute.org). Thus, a homozygous splice region variant in the PPP1R21 gene was identified . As expected, unaffected parents were heterozygous for the variant. Next generation sequencing results were confirmed by Sanger sequencing of an amplicon spanning the intron 8 and exon 9 junction of PPP1R21  absent in the control population , we found complete loss of protein expression on protein extracts from patient fibroblasts compared to matched controls  proteins were significantly dysregulated with a p value of < 0.05 with 584 proteins being up- and 269 proteins being downregulated, respectively , we identified 4801 proteins in the ely Fig. .Fig. 3PrOur proteomic data revealed an increase of proteins belonging to the ubiquitin–proteasome system including PSMB9, DTX3L, TRIM16, PSMG4, CRBN, TTC3, COMMD8, UBE2L6, PSME2, PSMD10, UBE2Z, PSMB10, AGAP3, RPS27A, UBE20, TRIM21, HECTD3, TMUB1, OBSL1, UBE2N, PSMG2, OS9, UBE2L3, CLU, UBXN7, USP7, SGTA, PSMA5, USP24, CUL7, BABAM1, and PSMD7 , the cerebellar degeneration-related protein 2-like , the neuronal calcium sensor 1 , and sacsin  are particularly interesting, as they are directly related to the neuronal system. Further upregulated proteins with known roles in neuronal or neuromuscular function and maintenance being include supervillin , hyccin (Q9BYI3), beta-1-synthropin (Q13884), neurabin-2 , and ataxin-10  Fig. .www.uniprot.org). The proteomic profiling data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD034993 and a list of all regulated proteins can be found in the supplement , filamin C , and tropomyosin  are notable. These proteins are crucial for intracellular integrity and stability as well as for muscle contraction, myogenesis, and maintenance of structural integrity of muscle fibers. Furthermore, tenascin-X (P22105) and fibulin-5 (Q9UBX5) show a 12.5-fold and a 6.6-fold downregulation, respectively. These proteins are responsible for structural assembly and stability by strengthening the binding between cells and with the extracellular matrix. Furthermore, we observed a profound decrease of proteins responsible for growth and formation of neural structures such as neurite extension, axon growth, and formation of neuronal circuits. These proteins include CD166 , glia-derived-nexin , and neuropilin1  , on signaling pathways , on RNA-processing (including splicing and translation), and on endocytosis and protein clearance (including proteasome). An impact on cell cycle and apoptosis is also indicated Fig. .Our proteomaps-based in silico analysis of cellular functions influenced by down-regulated proteins revealed an impact on metabolic processes , on cytoskeleton, on PI3K-AKT signaling as well as on ribosomal function Fig. .PPP1R21-mutant cells compared to controls  along with its co-chaperone GRP170 and further UPR-modulated proteins CALR, CALU, CANX, EDEM1, GRP94, PDIA2, PDIA3, and SEC63) and proteolysis  were performed. These studies revealed a clear effect of MG132 on expression levels of UPR-related genes in control fibroblasts, shown by significant increased expression of multiple genes after treatment . Additionally, we demonstrated that PPP1R21 patient fibroblasts showed a comparable expression level to basal control level without treatment, except for BiP and GRP170, which were significantly increased when compared to basal control levels. Treatment with MG132 decreased the expression of both transcripts of these two UPR factors, while other markers showed elevated levels after treatment . Activated autophagy after blocking of the proteasome by MG132 treatment is indicated by statistically significant increased expression of LC3 to similar extend in controls and patient-derived cells as well as by comparable increase of BECLN . BAG3 is decreased in patient derived but not in control fibroblasts after MG132 treatment. Proteasomal genes LMP7, PSME1, and PSME2 were highly increased after treatment in control, as well as patient fibroblasts but surprisingly not under basal conditions in patient-derived cells . This showed lower level of total ubiquitinated proteins under basal conditions in whole proteins extracts of fibroblasts derived from controls compared to the index patient, a finding which accords with the increased proteasomal activity in patient cells. After MG132-treatment, level of total ubiquitinated proteins is increased in both, patient-derived and control fibroblasts , indicating impaired actin remodeling in PPP1R21-mutant fibroblasts  in 18 control fibroblasts under basal conditions revealed an index comparable to the ones in patient-derived fibroblasts (0.516 and 0.561). After treatment of cells with MG132, no particular change was noticed in the control fibroblasts (0.516 versus 0.496) whereas in patient-derived cells, a slight increase was detected (0.561 versus 0.651). MG132-treated sts Fig. . Given tent Fig. .Fig. 6StPPP1R21 patient, harboring a homozygous splice variant (c.748-3A > G), providing associated phenotypical data and clinical comparisons with previously reported PPP1R21 patients. We also describe novel pathophysiological insights driven by the findings of our proteomic profiling on patient-derived fibroblasts.Here, we report on the first PPP1R21 gene  and belongs to a family of coiled-coil domain containing proteins, an evolutionarily conserved class of proteins with important roles in several developmental processes. Over the last three years, homozygous variants in PPP1R21 have been described in 11 patients, most of them sharing developmental delay/intellectual disability, hypotonia, distinctive facial features, and brain anomalies [The phosphatase 1 regulatory subunit 21 protein is encoded by the nomalies . In consnomalies –8, our pIn line with the known functions of PP1 as an important serine/threonine phosphatase essential for glycogen metabolism , alteredPPP1R21-mutant and control fibroblasts. These studies revealed a pronounced activation of the proteasome, as a multi-catalytic protease that degrades cellular proteins, even under treatment with MG132 in patient-derived cells. This finding is in line with our proteomic results showing a significant increase of a variety of proteins involved in the function of this protein clearance system as well as with the decrease of ubiquitinated proteins and LC3-I along with CD63 (two proteins involved in autophagy-related protein clearance) under basal conditions. Along this line, activation of the proteasome might reflect a cellular defense mechanism antagonizing the (massive) cytotoxic build-up of protein aggregates which have been identified with increased abundance in patient-derived fibroblasts by electron microscopy and immunofluorescence studies of p62. Hence, the co-occurrence of considerable elevated proteasomal activity and protein aggregates in fibroblasts of the PPP1R21-patient under basal conditions most likely correlates in terms of a cellular attempt to elevate protein clearance capacity to facilitate break-down of protein aggregates. Hereby, results of our combined transcript and proteomic studies indicate that activation of the proteasome in patient-derived fibroblasts is not related to an increased expression of proteasomal proteins (transcript level remain unchanged) and that their increased abundance might rather arise from altered protein stability toward forced proteasomal-based protein clearance. Results of our transcript studies revealed increased level of BiP and GRP170 encoding for a crucial chaperone system known to function as an analogous protein-disaggregation system activated under stress conditions in terms of a proteostasis-restorative ER-stress response [HSPB8. However, further proteomic data are not indicative for a broad activation of the ER-stress response, thus confirming the concept of protein aggregation arising beyond the protein production (within the ER). The UPS serves as the major protein quality control system in the eukaryotic cells, responsible for approximately 80–90% of the cellular protein degradation [Prompted by our proteomic findings indicative for activation of the ubiquitin–proteasome system, functional, transcriptional, and immunoblot studies were performed to investigate proteolysis in response . Chaperoradation . This prradation . Interesradation ) in PPP1radation . FurtherLC3 transcript level is increased, our immunoblot studies showed a reduced conversion of LC3-I to LC3-II in patient-derived cells treated with MG132 thus suggesting a partial impairment of autophagy-activation. This finding is supported by decrease of BAG3 in patient derived but not in control fibroblasts after MG132 treatment. Hence, our combined data suggest an impact of the activated protein clearance machinery on proper cytoskeleton which might impact on the regular transport of vesicles. However, further functional studies are needed to precisely decipher this molecular interplay in the context of PPP1R21 etiopathology.Notably, our MTT-assay revealed decreased cellular proliferation, an observation in agreement with the known function of PP1 in cell cycle regulation, accompanied with increased cellular toxicity burden. Moreover, a crosstalk between proteasome-mediated protein degradation and cytoskeleton has been described in the context of different studies  includinPPP1R21-patients. Given that expression level of sacsin was already linked to proper cytoskeleton and proteolysis [PPP1R21-mutant fibroblasts reflects the activation of cellular mechanisms to antagonize pathophysiological cascades initiated by the loss of functional PPP1R21. Increased abundance of supervillin might reflect the same hypothesis as loss of expression of functional supervillin was linked to the manifestation of myopathy characterized by myofibrillar disorganization and build-up of protein aggregates. Another interesting candidate in this context is neurabin-2: this protein also belongs to the PPP1R-protein family (PPP1R9B), modulates excitatory synaptic transmission and dendritic spine morphology, and operates in cross-lining of F-actin [Apart from indicating an activation of the ubiquitin–proteasome system, our proteomic study unraveled the dysregulation of various proteins known to play essential roles along the neuromuscular axis such as ataxin-10 and sacsin (SACS), both are linked to the manifestation of ataxia and cerebellar integrity. As mentioned above, our patient did not present with cerebellar abnormalities as observed in other teolysis , one mig F-actin . HoweverPPP1R21-patients are needed to generalize our findings and the related conclusions. Moreover, confirmational studies on cellular populations clearly vulnerable in PPP1R21opathy such as neurons are crucial to investigate the role of the neuronal relevant proteins in the molecular etiology of the disease. Organoids or animal models would be needed for this purpose. Further studies on other perturbed cellular processes such as metabolism are needed to address the overall pathophysiology of PPP1R21opathy.Although this study highlights the power of proteomics as an emerging tool for analysis of consequences of genetic variants even in individual patients suffering from nano-rare diseases, confirmational studies on fibroblasts derived from further In conclusion, our study extended the molecular genetic spectrum of PPP1R21opathy by describing the first splice variant leading to loss of protein expression and hereby described a patient presenting with a milder phenotype of NEDHFBA. Proteomic studies unraveled a dysregulation of various proteins playing crucial roles in proper function of the neuromuscular axis and increase of a variety of such might reflect the activation of cellular defense mechanisms. Our combined biochemical, microscopic, and cell biological investigations revealed increased presence of protein aggregates along with elevated cytotoxicity and activation of ubiquitin–proteasome system as well as altered cytoskeleton, a molecular crosstalk already described in the context of other diseases . Latter Supplementary file1 Dysregulated proteins their localizations, functions and associated diseases (XLSX 15 KB)Supplementary file2 Antibodies used in this study (DOCX 14 KB)Below is the link to the electronic supplementary material.


The application of integrated systems biology to the field of structural biology is a promising new direction, although it is still in the infant stages of development. Here we report the use of single particle cryo-EM to identify multiple proteins from three enriched heterogeneous fractions prepared from human liver mitochondrial lysate. We simultaneously identify and solve high-resolution structures of nine essential mitochondrial enzymes with key metabolic functions, including fatty acid catabolism, reactive oxidative species clearance, and amino acid metabolism. Our methodology also identified multiple distinct members of the acyl-CoA dehydrogenase family. This work highlights the potential of cryo-EM to explore tissue proteomics at the atomic level.  •Using cryo-EM to define the composition of a human mitochondrial lysate.•Simultaneously identifying and solving structures of nine mitochondrial enzymes.•Application of cryo-EM in studying high-resolution structural omics of human enzymes. et al use cryo-EM to simultaneously identify and solve structures of nine different enzymes from human liver mitochondrial lysate. Their work highlights the potential of this methodology in facilitating structural proteomics of a biological system at high resolution.Zhang  Mitochondria are double membraned organelles essential for a wide variety of functions. The most prominent role of mitochondria is the production of ATP, the major energy source of the cell . ATP is Recent advances in mass spectrometry have vastly expanded the large scale analysis of proteomes of human tissues . The conin silico purification. This methodology involves sorting images from a large and heterogeneous data set into different protein classes. The BaR platform includes preliminary 2D and 3D classifications and initial map constructions. Particle subsets with shared structural features are combined in order to create 3D ab initio templates. These initial templates are then used to extract additional particle views that share structural features, allowing for an expansion of the number of views in each protein class. The updated particle stacks are then processed using multiple rounds of 2D and 3D classifications in order to build high resolution maps for reviewing protein identities and solving final structural models. BaR is able to handle raw biological samples consisting of multiple proteins and enzymes, permitting us to extend the capacity of the structural tool of cryo-EM to study human tissues and organs in the context of systems biology.Recently, advancements in single-particle cryo-EM have facilitated the structural determination of traditionally challenging protein targets. Unlike mass spectrometry and proteomics, these structural tools typically require homogeneous and pure protein samples. This limits the ability to complement protein structure determination with existing systems biology approaches and explore native, heterogeneous samples. To address this challenge, our lab recently designed an approach to use cryo-EM to study tissue samples, called “Build and Retrieve” (BaR) . Specifivia the interconversion of aspartate and α-ketoglutarate to oxaloacetate and glutamate. Glutamate metabolism is regulated by GLUD1. Finally, SOD2, CAT, and ALDH2 are all involved in clearing toxic byproducts of oxidative respiration.To highlight the potential of the BaR methodology towards simultaneously elucidating structural information of a variety of mitochondrial proteins at near atomic resolutions, we enriched the protein component from human mitochondria lysate and then individually loaded the enriched protein samples onto cryo-EM grids for single particle imaging. Using this approach, we were able to simultaneously identify and solve cryo-EM structures of nine different human mitochondrial enzymes: short chain acyl-CoA dehydrogenase (SCAD), medium chain acyl-CoA dehydrogenase (MCAD), isovaleryl-CoA dehydrogenase (IVD), delta -delta  dienoyl-CoA isomerase (ECH1), aspartate aminotransferase (GOT2), glutamate dehydrogenase (GLUD1), mitochondrial superoxide dismutase 2 (SOD2), catalase (CAT), and mitochondrial aldehyde dehydrogenase (ALDH2). Functionally, the three acyl-CoA dehydrogenases—SCAD, MCAD and IVD—are involved in beta oxidation and amino acid catabolism. The ECH1 isomerase is a key enzyme in an auxiliary beta oxidation pathway for odd numbered unsaturated fatty acids. GOT2 facilitates recycling of NADH g. The extracted soluble lysate was then passed through a 0.22-μM filter and enriched using a Superdex 200 column  equilibrated with 20 mM Tris–HCl (pH 7.5) and 100 mM NaCl. We isolated three peaks based on the column separation. The first peak contained proteins ranging from 200 to 450 kDa including the identified ECH1 and GLUD1. Peak 2 contained proteins ranging in size from 150 to 250 kDa including the identified MCAD, IVD, SCAD, CAT, and ALDH2. Peaks 1 and 2 exhibited a degree of overlap. Our BaR methodology also identified CAT and ALDH2 subclasses in peak 1 but the particle stacks from peak 2 resulted in higher resolution maps. Lastly, peak 3 contained proteins smaller than 150 kDa including SOD2 and GOT2.Mitochondria isolated from liver tissue were purchased from Xenotech (BioIVT). The mitochondria were resuspended in a buffer containing 20 mM Tris–HCl (pH 7.5), 100 mM NaCl, and 5 mM Na-cholate. The soluble lysate fraction was separated from the membrane fraction by ultracentrifugation at 20,000Protein sizes corresponding to each peak were used for cryo-EM analysis. 3.5 μl (0.07–0.08 mg/ml) of each sample was applied to Quantifoil R 2/2 Cu 200 holey grids coated with graphene oxide. Samples were blotted for 8 s, then plunge frozen into liquid ethane using a Vitrobot (Thermo Fisher). The resulting grids were stored in liquid nitrogen until data collection.Samples from peak 1, peak 2, and peak 3 were collected at Pacific Northwest Cryo-EM Center using a Titan Krios cryo-electron transmission microscope (Thermo Fisher Scientific). Images were recorded using 1.0 to 2.5 μM defocus on a K3 direct electron detector (Gatan) using super resolution at 105,000× magnification. The super resolution size was 0.4125 Å/px resulting in a physical pixel size 0.825 Å/px. A second set of peak 3 samples was collected at the Cleveland Center for Membrane & Structural Biology using a Titan Krios cryo-electron transmission microscope (Thermo Fisher Scientific). Images were recorded using 1.0 to 2.5 μM defocus on a K3 direct electron detector (Gatan) using super resolution at 81,000× magnification. The super resolution size was 0.535 Å/px resulting in a physical pixel size 1.07 Å/px.via multiple rounds of 2D classification to identify particles with structural features. These particle stacks were then refined and grouped together to form initial 3D ab initio models. Initial models were built using C1 symmetry to reduce model bias. These models were then used as structural filters in a series of heterogeneous refinements. Each model “retrieves” additional particles relative to the initial stack, including a more diverse set of orientations. Refined particle stacks were then cleaned using 2D classification and used to generate refined 3D models. Non-uniform refinement was used to build the final maps. Once the high-resolution maps were built, the protein sequences were revealed using Deep Tracer  was predicted using the STRING database v.11.5 . The enz4HCO3 and 8 M urea. DTT was added to a final concentration of 10 mM at 25 °C for 30 min in order to reduce the sample. Alkylation was achieved using 25 mM iodoacetamide at 25 °C for 30 min. Samples were diluted by a factor of four using a digestion buffer containing 100 mM NH4HCO3 and trypsin/Lys-C mix (1:20 enzyme:substrate). Digestion took place overnight at 25 °C. The next day, digested peptides were desalted using a reverse-phase C18 Microspin column (Nest Group), washed two times with 150 μl aqueous solution containing 0.1% formic acid, and then eluted with 150 μl aqueous solution containing 80% acetonitrile and 0.1% formic acid.For each peak from the enrichment process using size-exclusion chromatography, 4 μg of sample was separately denatured in a buffer containing 50 mM NHLC-MS/MS was performed using a Thermo Scientific Fusion Lumos mass spectrometry system (Thermo Fisher Scientific). A Dionex 15 cm × 75 μm id Acclaim PepMap C18, 2 μm, 100 A reversed-phase capillary chromatography column was loaded with peptide. A linear gradient of acetonitrile (2–35%) in aqueous solution containing 0.1% formic acid was used for peptide chromatography for 90 min at a rate of 300 nl/min. The subsequent elution was introduced into the mass spectrometer operating in data-dependent MS to MS/MS switching mode with collision-induced dissociation mode. Full MS scanning was performed at 70,000 resolution between m/z = 350 and m/z = 1500. Protein identification was done by comparing experimental peptide MS/MS spectra to the UniProt human proteome database  using MaxQuant version 1.6.3.3 . While sin vitro proteomics approach to process the exact same samples used for single-particle cryo-EM data collection. Therefore, the peak 1, peak 2, and peak 3 mitochondrial lysate enriched from the Superdex 200 column  were individually aliquoted (2.0 μl from each lysate peak) and subjected to LC-MS/MS analysis . The signal intensity, protein sequence coverage, and protein accession number of the top 10 to 15 identified proteins of each sample are listed in via the PRIDE database.To confirm the presence of enzymes identified from BaR, we used Enrichment of the human liver mitochondrial lysate using size-exclusion chromatography resulted in three major protein peaks. The peak sizes corresponded to roughly 200 to 450 kDa, 150 to 250 kDa, and 80 to 150 kDa. We collected single particle cryo-EM images of each of the three peaks and processed them using the BaR methodology . The BaRTo identify whether these nine human mitochondrial enzymes interact and cooperate with each other, we constructed an interconnected network using the STRING database . We postAcyl-coenzyme A dehydrogenases are a family of enzymes involved in fatty acid oxidation and amino acid catabolism , 21. CurInitially, our BaR approach generated one structural class representative of the acyl-coenzyme A family. By layering an additional 3D classification step  into theSCAD gene can lead to SCAD deficiency (SCADD), a rare autosomal recessive disorder . Our structure is consistent with the previously determined crystal structure of rat SCAD (2VIG). The global fold of SCAD is a tetramer made up of a dimer of dimers. Each individual monomeric unit contains a β-sheet core flanked by N-terminal and C-terminal α-helical domains (C). The FAD cofactor-binding site is at the intersection of the three domains of each monomer (C). Additionally, the flavin ring interacts with the C-terminal domain of the corresponding subunit in each dimer set. The ligand-binding site lies parallel to cofactor and the catalytic site E392. Residues F152, L154, N207, F261, M265, L268, D269, R272, I342, I397, and the catalytic E392 residue form the substrate cavity (D). Interestingly, substrate specificity was altered in a mutagenesis study that altered this catalytic site to correspond to the catalytic site (E286) of IVD . Consistent with previous crystal structures of human (C). Residues T121, T124, A125, L128, F277, T280, R281, V284, Y398, Y400, and the catalytic residue E401 form the binding cavity (D). The substrate accommodation in the binding cavity is determined by a network of hydrogen bonds among residues Q120, T121, Q242, Y398, and Y400. In the MCAD structure, helix 4 and helix 6 are shifted relative to the corresponding SCAD structure. These two helices form the edge of the substrate cavity, resulting in a deeper substrate-binding pocket in the MCAD structure . Our structure is consistent with a previous crystal structure (C). The FAD cofactor-binding site is located in a position similar to that found in SCAD and MCAD. A key difference among these structures is the binding cavity, which is wider in IVD than in the SCAD and MCAD structures (A–C). This is due to the presence of a glycine at position G406 in the IVD molecule as opposed to the corresponding tryptophans Y391 (SCAD) and Y400 (MCAD), which allows for accommodation of the branched substrate. Additional residues that contribute to the binding cavity include L127, A131, L135, T200, L290, L402, Y403, G406, and A407 (D). A prior crystal structure also identified E286 as the catalytic base residue, in comparison to E401 in SCAD and MCAD . Our structure broadly agrees with the existing crystal structure of the rat enzyme (2VRE). The overall structure is a hexamer made up of a pair of trimers. Each monomer has two domains: an N-terminal catalytic domain and a C-terminal α-helical domain (C). Similar to other members of the isomerase family (D).A total of 8862 single particle cryo-EM projections were collected for this structure. From these projections, we constructed a high-resolution cryo-EM map of 2.96 Å and identified this protein as the ECH1 isomerase , A and Bt enzyme  and the l domain C. Similae family , the N-te family  identifie family D.Fig. 4CHumans have two aspartate aminotransferase isoenzymes, the cytosolic GOT1 and the mitochondrial GOT2 enzyme . These tA and B). The global structure of GOT2 is a homodimer, consisting of two 45-kDa monomers. Each subunit contains three main domains: a large domain that binds the pyridoxal phosphate (PLP) cofactor, a flexible small domain, and a helical bridge domain (B). The large PLP-binding domain consists of a seven stranded beta sheet core bracketed by sets of α helices. The active site is between the large and small domains and is supported by the large domain of the corresponding subunit. Residues S133, T135, H165, N215, D243, Y246, and K279 from one subunit as well as residues Y96′ and S317′ from the other subunit help form the cofactor and active site–binding cavity (C). A previous crystal structure also describes a small N-terminal domain (residues 3–14)   but our Glutamate dehydrogenase is a mitochondrial matrix protein expressed in high levels in the liver, brain, kidney, heart, pancreas, ovaries, and testes . FunctioA and B). In agreement with previous human (C). The GTP-binding site is bracketed by several key basic residues, including H266, R274, R318, R322, and H507, as well additional residues I269, S270, and Y319 (D). There is a large degree of conformational flexibility in both the NADH-binding domain and antenna domain, particularly in the unbound state . The global architecture of SOD2 is a homotetramer formed by a dimer of dimers. Similarly to existing crystal structures (C). A central cavity is formed by the interface of each subunit’s β-sheet core, while dimer interfaces consist of α-helical bundles from the N-terminal domains. Elements of both domains contribute to the Mn2+-binding site for each subunit. Specifically, this cavity is formed by the H50 and H98 from the N-terminal domain and D183 and H187 from the C-terminal domain. An additional stabilizing hydrophobic pocket is formed by residues H54, F101, W102, W147, and W185 (D).Our BaR methodology classified a total of 50,014 single particle projections for the SOD2 class. Based on these projections, we constructed a high-resolution density map of 2.91 Å , A and Bructures , 48, eacructures C. A centand W185 D.Fig. 7C2O2, is a toxic byproduct of many metabolic pathways. CAT is an enzyme found in many organs whose main function is to break down hydrogen peroxide into water and oxygen to prevent H2O2-induced damage . Consistent with existing crystal structures of liver CAT (C). The hydrophobic core of the subunit is made up of a β-barrel domain made of eight antiparallel β sheets. Eight α helices, including four C-terminus helices and four located between β sheets β4 and β5, form the α-helical domain of each subunit. Two subunits are linked by threading the N-terminal arm of each subunit through the wrapping loop domain of the other subunit. Two sets of linked subunits assemble to form the tetramer in a manner where the N-terminal arms cover the heme active sites. A long channel in the β barrel domain leads to each of the heme active site, which consists of the residues M61, R72, V73, V74, R112, V146, N148, P158, F161, S217, H218, F334, M350, R354, A357, Y358, T361, H362, and R365 (D). The NADPH-binding site rests between the α-helical and β-barrel domains and is stabilized by residues H194, F198, S201, R203, N213, K237, Q282, V302, H305, Q442, F446, V450, and L451 (E).We collected a total of 31,971 single-particle cryo-EM projections for this class of protein images. The BaR methodology allowed us to construct a high-resolution cryo-EM map. Subsequently, we were able to identify this protein as the CAT enzyme and resolve its structure to a resolution of 2.58 Å , A and Biver CAT , each CAiver CAT C. The hyand R365 D. The NAand L451 E.Fig. 8Cm for the cofactor NADH. Recently, this ALDH2 mutation has been reported as a risk factor for late-onset Alzheimer’s disease, potentially via a buildup of reactive oxidative species . The structure of ALDH2 is a dimer of dimers, forming a functional tetramer.We collected a total of 82,360 single-particle cryo-EM projections for this class of protein images. Based on these projections, the BaR methodology allowed us to construct a high-resolution cryo-EM map. Subsequently, we were able to identify this protein as the ALDH2 enzyme and resolve its structure to a resolution of 2.66 Å , A and BC). The coenzyme domain (24–155 and 176–287) possesses an NADH-binding site. Both the cofactor and catalytic domain (residues 288–502) contain a five stranded α/β sheet followed by two antiparallel β strands. The oligomerization domain (156–175 and 503–511) is a three stranded antiparallel β sheet. Presumably, residues W185, N186, K209, E212, S263, Q366, E285, and C319 are engaged in NADH binding and C319 is the catalytic residue . However, we did not observe bound NADH in our cryo-EM structure.Matching with existing crystal structures  of the AFig. 9Cellular processes are made up of a complex concert between protein, small molecule, and enzyme interactions. A comprehensive systems biology approach is highly desirable at the structural level, as it offers a holistic understanding of the underlying biological interactions. We previously developed a BaR methodology  in orderAs with previous BaR applications , 56, 57,3 of Escherichia coli and the monomeric HpnN transporter of Burkholderia pseudomallei  and less abundant (<10%) unidentified proteins within a heterogeneous protein mixture sample. Nonetheless, our application of BaR on soluble mitochondrial organelles is only a small slice of the potential systems biology applications and there are several limitations in this methodology. First, proteins with cryo-EM maps of >4.0 Å remain difficult to identify. Second, it is difficult, if not impossible, to identify a particular protein if the particle population of the protein is <5% in the heterogeneous sample. In addition, preferential orientation of protein images can be a significant problem, as BaR may not be capable of finding a solution for a particle class with insufficient orientation views. Indeed, in our study, there were several unique structures that we were unable to identify. This is most likely the result of preferential orientation, which limits the ability to build an initial starting model and affects the downstream resolution.via mass spectrometry and cryo-EM single particle counts. This is surprising as both BaR and LC-MS/MS are based on analyzing and counting protein populations. However, this discrepancy could be easily explained. It could be attributed to additional steps for the preparation of cryo-EM grids for single-particle imaging. As an example, the procedures for preparing a cryo-EM grid include a blotting process, where the filter paper may have different absorptivity for different proteins. During blotting, a large population of proteins may be absorbed by the filter paper, thus significantly altering the composition of protein components and their relative populations on the grid.It is interesting to note that we did not observe a strong correlation between the population ranking of proteins In this study, we only describe structures from healthy tissue. Given the prominent role of our subset of identified enzymes in metabolic disorders, this offers an exciting potential of using BaR to compare healthy and mitochondria from different disease phenotypes. This approach could help uncover salient information regarding the mechanism of a particular disease. In addition, our mitochondria were derived from the liver. Comparing mitochondria from different tissue types can possibly be another research approach that will complement existing proteomics methodologies.E. coli K12 cell membrane  via the PRIDE partner repository (Coordinates and EM maps for SCAD, MCAD, IVD, ECH1, GOT2, GLUD1, SOD2, CAT, and ALDH2 can be found at PDB accession numbers This article contains All authors declare that they have no competing interests.


Microglia possess the unique ability to migrate in the mature brain, but the factors that govern this are poorly understood. Here, we show that while the population of mobile (“patrolling”) microglia is small under normal conditions, it can be rapidly expanded following injury. Microglia mobility after injury was dependent on biological sex, such that microglia from male mice were more likely to migrate, and they migrated greater distances toward the site of injury compared to their female counterparts. This migratory ability was regulated by interferon gamma signaling in males, but not females. Our findings highlight the diversity of factors that regulate microglia migration and add to emerging data that male and female brains have different immunological responses to injury. Although microglia possess the unique ability to migrate, whether mobility is evident in all microglia, is sex dependent, and what molecular mechanisms drive this, is not well understood in the adult brain. Using longitudinal in vivo two-photon imaging of sparsely labeled microglia, we find a relatively small population of microglia (~5%) are mobile under normal conditions. Following injury (microbleed), the fraction of mobile microglia increased in a sex-dependent manner, with male microglia migrating significantly greater distances toward the microbleed relative to their female counterparts. To understand the signaling pathways involved, we interrogated the role of interferon gamma (IFNγ). Our data show that in male mice, stimulating microglia with IFNγ promotes migration whereas inhibiting IFNγ receptor 1 signaling inhibits them. By contrast, female microglia were generally unaffected by these manipulations. These findings highlight the diversity of microglia migratory responses to injury, its dependence on sex and the signaling mechanisms that modulate this behavior. Microglia are highly dynamic, innate immune cells that reside within the brain –3. Micro1215The mobility of microglia toward sites of injury or toxic proteins in neurodegenerative disease, could be important for mollifying toxicity and resolving injury –24 but c26Although we have a better understanding of the molecular mechanisms that regulate microglial surveillance and process chemotaxis , 35, the42Despite our increased understanding of microglial biology , importaSI Appendix, Fig. S1). Since fully differentiated neurons in the adult mouse cortex do not migrate, this allowed us to estimate measurement error in vivo. By tracking 206 neurons in three mice, we found the average displacement of cells was 1.84 ± 1.45 µm for 0 to 12 h and 1.61 ± 1.53 µm for 12 to 24 h . To minimize the possibility of false positive detection of cell “movement”, we determined that any distance ≥7.46 µm per 12-h period (four SD above mean distance derived from both 12-h timepoints) represented true cell movement. This threshold formed the basis for what we defined as a “mobile” cell.For the sake of clarity, the terms microglial “mobility or movements” herein refers to the movements of microglia cell bodies to new positions, not the movement of processes. To quantify microglial mobility in vivo, we had to first define what distance would constitute a true movement. Cells within the living brain can be pushed or displaced small distances for a number of reasons  and repeated 3-dimensional measurements possess an intrinsic degree of error. Therefore, we measured the distance between GFP-labeled VIP interneurons in the cortex and a fiducial landmark at 12-h intervals in mice lightly anesthetized with isoflurane . Unfortunately, because the density of cell labeling was high, the shape of cells changed constantly and both microglia and macrophages express eGFP; we could not be sure if cells that changed (say appeared or disappeared) were microglia that moved a long distance or perhaps were infiltrative macrophages. Furthermore, we could not track single-cell movements after injury when cells rapidly cluster around a focal injury site .To track the migration of microglia in adult mice, we first utilized heterozygous Cx3cr1-eGFP reporter mice , as descTo track the movements of individual cells, we crossed the tamoxifen inducible and microglia-specific Tmem119-CreERT2 mouse line with cre-dependent tdTomato reporter mice (Ai9). Consistent with its original description , low-dosLeft, two-tailed t test, t(14) = 0.59, P = 0.57) or per mouse  = 1.48, P = 0.20). Stratifying the movement of these mobile cells based on sex, did not reveal a significant effect of time or sex  = 0.39, P = 0.54, Sex: F = 4.09, P = 0.053), although there was a trend toward reduced mobility in female cells . These results indicate that a small population of microglia are actively moving about in the uninjured brain.Having optimized methodologies to track microglia in vivo, we then imaged their mobility over 1 d at time 0, 12, and 24 h B. This iP < 0.01). However, to our surprise, the absolute distance of migration  = 15.6, P = 0.0001; Time: F = 0.81, P = 0.37; Sex x Time: F = 6.02, P = 0.02). Furthermore, microglia in male mice showed significantly greater net movement toward the CMB than female microglia  = 25.3, P < 0.0001; Time: F = 1.21, P = 0.27; Sex x Time: F = 1.99, P = 0.16). On average, male microglia migrated in a directional manner toward the microbleed  whereas female cells showed little directional movement (P > 0.05 for one sample t tests conducted at each time point). The fraction of mobile microglia in male mice was significantly greater than in female mice (P < 0.01). And finally, as it was plausible that a cells’ relative distance from the CMB could influence mobility, we found in male mice that microglia closer to the bleed were more likely to migrate , whereas in female mice, this relationship disappeared . We should note that the size of the bleed, based on the area of dye extravasation, was not significantly different between sexes  = 0.93, P = 0.33). In addition to migratory behavior after injury , we also noted possible examples of microglia proliferation , although these were very infrequent events. Collectively, these results indicate that sex has a strong influence on mobility after vascular injury.Since previous reports have shown that male microglia have a higher capacity to respond to ATP signals, display a more proinflammatory pattern of gene expression, and can appear morphologically different than females –46, we tSI Appendix, Fig. S6A), nor was there any significant relationship between movement distance and depth, even when stratifying cells by sex . Given there is growing appreciation for the diversity of microglia, especially those that reside along capillaries (referred to as capillary associated microglia or “CAM”) that modulate blood flow . Separating our analysis based on sex did not reveal any significant differences in movement between CAM vs. Non-CAM . In summary, our findings indicate that a cell’s depth below the cortical surface or whether it was associated with a capillary, does not significantly influence mobility.Given that sex and distance from injury were not the only variables at play in these experiments, we examined other factors that could influence mobility. First, we considered a cell’s depth from the cortical surface. Parsing microglia based on whether they were stationary or mobile did not reveal any systematic difference in cortical depth . To knock down IFNγ signaling specifically within microglia, we crossed the Ai9:Tmem119cre line with IFNγ receptor 1 floxed mice (Ifngr1fl/fl), which previous studies have used to induce cell-specific knockdown of IFNγ signaling  = 6.64, P = 0.01, Bleed: F = 22.16, P < 0.0001, IFNγ: F = 7.613, P < 0.001), with strong trends for a Bleed x IFNγ  = 2.56, P = 0.08) and Sex x IFNγ  = 2.74, P = 0.07) interaction. Under normal conditions , we did not find a single mobile microglia in either male or female mice with cre-recombinase-dependent knockdown of Ifngr1  in the % mobile microglia as a fraction of total cells sampled or % cells per mouse , whereas a moderate relationship was evident in WT female mice injected with IFNγ (2 = 0.19). Lastly, we plotted the absolute and net movement of all mobile microglia in each group . However, there was a significant main effect of microbleed on the net movements of microglia at 12 to 24 h  = 6.43, P = 0.01), as well as the sum of absolute  = 7.19, P < 0.01) and net  = 6.89, P < 0.01) movements . This finding suggests that IFNγ signaling may exert some tonic effect on mobility in the normal brain, perhaps through low-level expression of IFNγ in resident immune cells, or conceivably constitutive IFNγ receptor activity. Consistent with this idea, the Kipnis group , our results showing sex differences in migratory responses to injury could have functional implications. Human studies are rather mixed but show that males diagnosed with cerebral amyloid angiopathy , mild co+/GFP mice (JAX# 005582) on a C57BL/6J background (JAX# 000664). For cre-dependent inducible expression of the tdTomato reporter and/or knockdown of Ifngr1, we utilized Ai9 reporter mice (B6.Cg-tm9(CAG-tdTomato)HzeGt(ROSA)26Sor/J, JAX# 007909) crossed with an inducible microglia-specific cre driver line (em1(cre/ERT2)GfngTmem119/J, JAX# 031820). These Ai9:Tmem119CreERT2 mice were then crossed with floxed Ifngr1 mice  and bred to homozygosity. All mice were housed in groups on a 12-h light/dark cycle in ventilated racks in a humidity (RH 40 to 55%) and temperature-controlled room (21 to 23 °C). Mice were provided food and water ad libitum. All experiments comply with the guidelines set by the Canadian Council on Animal Care and approved by the local university Animal Care Committee. Reporting of this work complies with ARRIVE guidelines.Adult male and female mice between 2 and 6 mo of age were used in this study. For labeling of microglia and monocyte-derived macrophages, experiments involved heterozygous CX3CR12, 20% O2) at a flow rate of 0.7 L/min. A temperature feedback regulator and rectal probe thermometer maintained animals’ body temperature at 37 °C throughout the procedure. After subcutaneous injection of lidocaine under the scalp, an incision was made along the midline. A custom metal ring  was positioned over the right somatosensory cortex and secured to the skull with metabond adhesive. A circular area (diameter of ~4 to 5 mm) of skull within the metal ring was thinned using a high-speed dental drill, and ice-cold HEPES-buffered artificial cerebrospinal fluid (ACSF) was periodically applied to the skull for cooling purposes. Fine forceps were used to remove the thinned piece of skull and the exposed brain was kept moist with gel foam soaked in cold ACSF. A 5- or 6-mm coverslip (no. 1 thickness) was placed over the exposed brain and secured to the skull using cyanoacrylate adhesive. After the procedure, mice were injected with 0.03 mL 2% dexamethasone (i.p.) to reduce acute inflammation resulting from the procedure. Mice were monitored while they recovered under a heat lamp and then were returned to their home cage.Mice of at least 2 mo of age, were anesthetized using isoflurane (2% for induction and 1.3% for maintenance) in medical air  dissolved in corn oil (Sigma #T5648). If mice failed to show sufficient reporter expression 2 wk later, they were reinjected with the same dose of tamoxifen.Two to four wk after cranial window implantation, cre-recombinase-dependent expression of tdTomato reporter and/or knockdown of For two-photon imaging, mice were lightly anesthetized with isoflurane , and then received 0.1 mL of either 1.5 to 3% fluorescein isothiocyanate–dextran (FITC) or Texas Red dextran , to permit visualization of the cerebral vasculature. The metal ring on the mouse’s head was fixed into a custom imaging stage. High-resolution two-photon image stacks of the vasculature and GFP or tdTomato expressing microglia/macrophages were acquired in vivo using an Olympus FV1000MPE laser scanning microscope fed by a mode-locked Ti:Sapphire laser source  and equipped with a water-dipping 20× objective lens . The laser was tuned to 900 nm for imaging Texas Red dextran and eGFP, while 945 nm was used for imaging FITC and tdTomato-labeled microglia. Emitted light was split by a dichroic filter (552 nm) before it was directed through band-pass filters (495 to 540 nm and 558 to 630 nm). Images were collected at 1.75-μm z-step intervals from the cortical surface to a depth of 75 to 200 µm, with each image covering an area of 635.3 × 635.3 μm (1024 × 1024 pixel sampling). In order to maximize sampling of sparsely labeled microglia, we typically imaged 2 to 5 different regions within each mouse, spaced at least 500 µm apart from each other. Capillaries that were 3 to 6 µm in diameter were targeted for laser-induced rupture. As previously described, we raster scanned a 4x4-µm region of interest centered on the capillary for 3 to 8 s using the femtosecond laser . The rupture of a cortical capillary was confirmed in real-time by the extravasation of fluorescently labeled blood plasma. Brightfield images of the brain’s surface and vascular landmarks were used to relocate imaging areas over time.t test, t(5) = 0.75, P = 0.48; ABS movement Sum: two-tailed t test, t(30) = 0.89, P = 0.38), therefore data from these groups were pooled together under “WT bleed” group.For modulating IFNγ signaling in vivo, we used two approaches. First for stimulating IFNγ signaling , we randtm1(cre)ZjhVip/J, JAX# 010908) at 12-h intervals . Using Neurolucida software, we quantified the Euclidean distance from the center of each cell body to a fiducial vascular landmark in the center of the image. Performing this analysis in three mice with 206 neurons, yielded a mean error of 1.84 ± 1.45 µm and 1.61 ± 1.53 µm for 0 to 12 and 12 to 24-h measurements. To minimize any false-positive errors, we set the minimum migration cutoff at 4-SD above the mean error values to yield a minimum cutoff migration distance of 7.46 µm in a 12-h period.In order to estimate error associated with three-dimensional measurements in vivo, we repeatedly imaged GFP-labeled VIP neurons in mice  changed by more than 7.46 µm at 0 to 12 or 12 to 24 h. The sum of mobile cells represented all those that exceeded this criteria at either or both time intervals. Since cells could move toward or away from a vascular landmark or bleed site, movement was calculated as either the absolute value (thus indifferent to direction) or the net value relative to vascular landmark or bleed location. For the analysis of movement in CAM, we first determined whether the closest edge of a microglia soma was within 1.86 µm (three pixels) from the lumen of a flowing capillary or not. Based on this dichotomy, we than analyzed the absolute and net movement of each microglia across and within sexes. In order to estimate the extent of plasma extravasation after microbleed, we calculated extravasation area from single images collected immediately after the rupture that were then mean filtered (radius = two pixels) and thresholded using Huang’s method in Image J.P < 0.05, **P < 0.01, ***P < 0.001.Absolute and net distances traveled by mobile microglia were analyzed with two-way or three-way ANOVA using either time, injury (bleed vs. no bleed), Sex and IFNγ status (WT control vs. WT+IFNγ vs. Ifngr1 KD) as factors. Post hoc tests were adjusted using Sidak’s multiple comparisons tests. Chi-squared analysis was used to determine if the fraction of mobile cells differed between experimental groups. The “observed” values for each experimental group were compared to the “expected” values, which were derived from no bleed or male groups in Appendix 01 (PDF)Click here for additional data file.


M. luteus and B. subtilis) and Gram-negative bacteria (E. coli and S. typhimurium). Three polymers, poly, poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride), and poly(vinyl benzyl trimethylammonium chloride), were examined to explore the effect of molecular weight  on their antimicrobial activity and toxicity to mammalian cells. The mechanisms of action of the polymers were investigated with dye-based assays, while Scanning Electron Microscopy (SEM) showed collapsed and fused bacterial morphologies due to the interactions between the polymers and components of the bacterial cell envelope, with some polymers proving to be bactericidal and others bacteriostatic, while being non-hemolytic. Among all the homopolymers, the most active, non-Gram-specific polymer was poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride), with a molecular weight of 40 kDa, with minimum inhibitory concentrations between 16 and 64 µg/mL, showing a bactericidal mode of action mediated by disruption of the cytoplasmic membrane. This homopolymer could be useful in biomedical applications such as surface dressings and in areas such as eye infections.Ammonium group containing polymers possess inherent antimicrobial properties, effectively eliminating or preventing infections caused by harmful microorganisms. Here, homopolymers based on monomers containing ammonium groups were synthesized via Reversible Addition Fragmentation Chain Transfer Polymerization (RAFT) and evaluated as potential antibacterial agents. The antimicrobial activity was evaluated against Gram-positive ( E. coli, S. aureus, and K. pneumonia) trimethylammonium chloride) and poly(vinyl benzyl trimethylammonium chloride) were also selected, to investigate the effect of molecular weight  on their antimicrobial activity and mechanisms of action. In this study, we focused on ammonium group containing homopolymers synthesized via RAFT polymerization. Specifically, we explored the impact of molecular weight on the antimicrobial activity, cytotoxicity, and biocompatibility, as well as their antimicrobial mode of action. A key advantage of these polymers is the fact that there is no need for modification, such as quaternization which typically requires alkylating agents to drive full functionalization. At the outset, five different monomers were polymerized via RAFT polymerization to give 20 kDa polymers, and the antimicrobial activity of the homopolymers was analyzed on Gram-negative ethyl]trimethylammonium chloride in water with 4-cyano-4-[(dodecyl sulfanyl thiocarbonyl)sulfanyl]pentanoic acid) (CTA1) was not achieved due to the limited solubility of CTA1 in aqueous environments. Water was the best solvent for the chain transfer agent 4-cyano-4-(phenylcarbonothioylthio) pentanoic acid (CTA2) with [2-(methacryloyloxy)ethyl]trimethylammonium chloride and gave the polymer in 5 h with 95% monomer conversion. GPC analysis showed a narrow, unimodal peak for the polymers . It is worth noting that this is the first reported homopolymer synthesis of poly [2-(methacryloyloxy)ethyl]trimethylammonium chloride (PMEATCL) in the presence of the chain transfer agent 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid and the water-soluble azo initiator 4,4-azobis . Similarly, the polymerization of vinyl benzyl trimethylammonium chloride was achieved with high conversion of the monomer to the polymer under similar conditions . Meanwhile, 10 and 20 kDa polymers of PVMBT and PMETACL were synthesized by changing the [chain transfer agent]/[initiator] ratios. The polymerization of 3-[(methacryloylamino)propyl]trimethylammonium chloride was carried out under similar conditions; however, only 44% conversion was obtained after 24 h reaction (giving a polymer of approximately 20 kDa). GPC analysis of PVMBT and PMET3 showed polymers smaller than those calculated, perhaps explained by the structural differences in these homopolymers and the standard polymers used in GPC calibration (polyethylene glycols). The RAFT polymerization of diallyldimethylammonium chloride was challenging due to poor monomer reactivity; indeed, monomers such diallyldimethylammonium chloride are typically classified as “less activated” and require xanthate- or dithiocarbamate-based chain transfer agents . Thus, tB. subtilis, E. coli) and Gram-positive  bacteria by the resazurin-based microtiter viability assay. The blue dye, resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide), can be irreversibly reduced to the pink, and highly red fluorescent, resorufin, by oxidoreductase within viable cells . Briefly, serially diluted polymers were added to bacterial cultures to assess the concentration at which bacterial growth ceased. Color changes were observed visibly and spectrometrically. All assays were carried out in triplicate and the results of this are shown in The minimum inhibitory concentrations (MIC) of the polymers were determined against Gram-negative , medium (20 kDa), and high (40 kDa) molecular weights of a particular polymer at three different concentrations were evaluated see  and showAmong the tested polymers, PDADMAC-40 showed the best inhibition of all bacteria strains, with an MIC in the range of 0.43–0.86 µM (16–32 µg/mL). However, it is worth mentioning that increased polymer chain length was also found to lead to enhanced cytotoxicity, with PDADMAC-40 being highly cytotoxic to HeLa cells even at the lowest concentration (1.25 µM) tested. Importantly, PDADMAC-20, PMETACL-40, and PVMBT-40 showed similar MIC values (0.9 µM), but were much less cytotoxic.A live/dead assay was used for the assessment of bacterial membrane integrity after treatment with the polymers, with double staining using SYTO 9 and propidium iodide (PI). E. coli and M. luteus were treated with the most active polymers  and analyzed by SEM to observe the bacterial morphology. As shown in E. coli “outer envelope” showed wrinkles and deep hollows, while a misshaped and raptured membrane of M. luteus was observed. Moreover, pores were found on the surface of M. luteus, suggesting the permeabilization of the plasma membrane and leakage of cellular content. These phenomena suggest that these polymers kill bacteria by destroying their cell membranes.In order to understand the mechanism of action, E. coli was depolarized by the polymers, with PVMBT-40 giving a 10-fold increase in fluorescence (B. subtilis) was measured by the polymer-induced leakage of the fluorescent dye calcein. Analysis (v/v), with PDADMAC-20 acting faster than PMEATCL-40, presumably due to its lower molecular weight. The more hydrophilic polymer chains of PMEATCL-40 penetrated into the peptidoglycan layer more efficiently than PVMBT-40.Perturbation of the bacterial outer membrane (Gram-negative bacteria) was assessed using the fluorescent probe (1-N-phenyl-naphthylamine) (NPN) that enters and binds damaged membranes. Supported by the SEM images, the assay results suggested that the outer membrane of rescence . PresumaAnalysis  showed tAs shown above, the higher-molecular-weight polymers showed better antimicrobial activity. An MTT cytotoxicity assay showed that the homopolymers see  displayeThe hemolytic activity was investigated as a biocompatibility indicator  were purchased from Sigma Aldrich , Fluorochem , Fisher Scientific , or Alfa Easer  and used as received, unless otherwise noted. Except for diallyldimethylammonium chloride (65% wt. in H2O), all monomers were passed through a basic alumina column to remove inhibitors before polymerization reactions. The chain transfer agent, S-ethoxythiocarbonyl mercaptoacetic acid (CTA3), was synthesized according to the literature pentanoic acid (CTA1), 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CTA2), NaOH, anhydrous acetonitrile, ethanol, acetone, hexane, and deuterated solvents (Dterature . Deioniz1H NMR spectrometry in D2O or CDCl3). The experimental molecular weights of the polymers were calculated based on the integration of the disappearing vinyl protons of the responsible monomer and the appearance of the methylene peak of the formed polymer via 1H NMR. Theoretical molecular weights were calculated according to the literature 0 are the initial concentrations of the monomer and chain transfer agent; p is the monomer conversion; and MwM and MwCTA are the molar masses of the monomer and chain transfer agent, respectively.Monomers were subjected to free radical polymerization to determine the reaction conditions and understand the compatibility of monomers, initiators, and solvent. Subsequently, all monomers were subjected to RAFT polymerization with a suitable chain transfer agent. During the polymerization reactions, aliquots were taken to follow monomer conversion  dihydrochloride (AAPH) and the xanthate-type chain transfer agent S-ethoxythiocarbonyl mercaptoacetic acid (CTA3) at 60 °C. The other monomers were polymerized using the initiator 4,4-azobis  (ACVA) and either 4-cyano-4-(phenylcarbonothioylthio) pentanoic acid (CTA2) or 4-cyano-4-[(dodecyl sulfanyl thiocarbonyl)sulfanyl]pentanoic acid) (CTA1) at 70 °C as the chain transfer agents. In order to synthesize the desired different molecular weights of the polymers, the chain transfer and initiator concentrations in the reaction were tuned while the monomer concentrations were kept constant .First, 2-(dimethylamino)ethyl methacrylate (5 mL), radical initiator 4,4′-azobis(4-cyanopentanoic acid) (ACVA), (0.0084 g), chain transfer agent CTA1-(4-cyano-4-[(dodecylsulfanyl thiocarbonyl)sulfanyl] pentanoic acid) (0.0726 g), and 10 mL ethanol were mixed in a septum sealable glass tube (50 mL). The solution was degassed by argon bubbling for 30 min. The polymerization was carried out at 70 °C for 24 h. The polymer was purified by removal of the ethanol, redissolution in THF, and precipitation using n-hexane. The polymer was dried under vacuum at 40 °C for 2 days to give a yellow-colored polymer (78% yield).2O), initiator 4,4-azobis , raft agent CTA2 (40 mg), and water (17.9 mL) were mixed in a 50 mL Schlenk flask and the reaction solution was purged with nitrogen for 30 min. The polymerization reaction was carried out at 70 °C for 24 h. Aliquots were taken at different time intervals to check monomer to polymer conversion via 1H NMR analysis. The resulting polymer was purified by precipitation using acetone, redissolved in methanol, and again precipitated using acetone and dried under vacuum at 40 °C to give a salmon-colored polymer (94% yield). This polymer was synthesized with three different molecular weights by changing the chain transfer agent to initiator ratio. First, [2-(methacryloyloxy)ethyl]trimethylammonium chloride) , initiator 4,4-azobis, CTA2 (40 mg), and water (17.5 mL) were mixed in a 100 mL flask and the reaction solution was purged with nitrogen for a 30 min. It was then placed into a preheated oil bath at 70 °C for 24 h. The resulting polymer was purified by precipitation with excess acetone, redissolved in methanol, re-precipitated with acetone, and dried under vacuum at 40 °C (51% yield).First, [3-(methacryloylamino)propyl]trimethylammonium chloride (13 g 50% wt in HThe monomer (4 g), 4,4-azobis (ACVA) as an initiator (7.84 mg), CTA2 (28 mg), and water (25 mL) were mixed in a 50 mL Schlenk flask and purged with nitrogen for 30 min. The polymerization reaction was carried out at 70 °C for 24 h. The resulting polymer was purified by precipitation using THF and dried under vacuum at 40 °C (89% yield). This polymer was synthesized with three different molecular weights by changing the changing the chain transfer agent to initiator ratio.2O), initiator 2,2′-azobis (2-methylpropionamidine)dihydrochloride (AAPH) (0.019 g), CTA3 (0.041 g), and water (8 mL) were mixed in a 50 mL Schlenk tube. Then, the reaction mixture was degassed by bubbling with argon for 30 min and placed in preheated oil bath at 60 °C for 24 h. The resulting polymer was precipitated in a mixture of acetone/ethanol (1:1) three times and dried under vacuum at 40 °C (70% yield). This polymer was synthesized with three different molecular weights by changing the chain transfer agent to initiator ratio.The polymerization of diallyldimethyl ammonium chloride was carried out by the protocol of Demarteau . DiallylThe synthesized polymers were numbered as follows: PDMAEMA (P1), PMETACL (P2), PMET3 (P3), PVBMT (P4), and PDADMAC (P5).1H NMR  δ 4.63 , 3.93 , 1.34 . 13C NMR  δ 213.95, 172.80, 71.72, 37.32, 12.79)  . LC-MS (terature .3 or D2O at either 500 MHz (H1 NMR) or 125 MHz (C13).All monomers, polymers, and RAFT agents were analyzed on a Bruker AVA500 spectrometer in CDCl2PO4, at pH 2.5, with a flow rate of 1.0 mL min−1 at 25 °C. Calibration was achieved using InfinityLab EasiVial poly(ethylene oxide) standards with Mn values ranging from 1.1 to 905 kDa. GPC analysis of poly (2-(dimethylamino)ethyl methacrylate) (PDMAEMA) was carried out on an Agilent 1260 Infinity system on two PL-GEL mixed-c columns (5 μm) with both UV and RI detectors. The eluent used was THF at a flow rate of 1.0 mL min−1 at 35 °C. Molecular weights were obtained relative to poly(methyl methacrylate) standards.Aqueous-based GPC analysis of the polymers was carried out on an Agilent Technologies 1100 system, 8 μm Agilent PL Aquagel-OH 30, and 8 μm Agilent PL Aquagel-OH 40 columns with an RI detector. The eluent was 0.50 M acetic acid, 0.30 M NaHEscherichia coli, Salmonella typhimurium, Micrococcus luteus, and Bacillus subtilis) was streaked onto Luria–Bertani, nutrient agar, tryptic soy agar, and nutrient agar plates, respectively. A single bacterial colony was transferred to 5 mL of the respective broth medium and incubated at 37 °C and grown until the mid-log phase. The culture was diluted and the concentration adjusted to 0.5 McFarland standard with sterile Mueller–Hinton broth (2 × 107 CFU/mL). The resazurin solution was prepared in a brown glass vial by dissolving 34 mg of resazurin in sterile distilled water (5 mL) with vortexing for 1 h to ensure homogeneity. A single 96-well microtiter plate  was dedicated to each bacterial species to prevent contamination. The design of the assay was prepared with two rows and two columns from each end filled with sterile water to avoid edge effects. The assay was adapted from previously reported protocols [typhimurium), gentamicin (for E. coli and M. luteus), and clindamycin (for B. subtilis). Five μL of the diluted bacterial suspension (2 × 107 CFU/mL) was added into all wells (except the broth sterility and polymer sterility control columns) and mixed thoroughly. After overnight incubation at 37 °C, resazurin solution  was added to all wells and incubated at 37 °C for another 4 h. Changes in color from blue  to pink  were recorded at 595 nm on a microplate reader (BioTek Synergy HT). The lowest concentration that did not show a color change was considered as the minimum inhibitory concentration (MIC). Each assay was performed in triplicate.Antibacterial activity was assessed using a resazurin colorimetric assay . Five µLrotocols . Two colIn order to determine the effect of the molecular weight on the antibacterial activity, three differently sized polymers were synthesized for PDADMAC, PMETACL, and PVBMT.Zone diffusion assays and growth kinetics: Agar plates were inoculated by spreading 100 µL bacteria (1 × 106 CFU/mL), 1-cm-diameter wells were punched into the agar, and 100 µL of polymer solution (at 2× MIC) was added into the wells and the zones of inhibition were measured after 8 h.Bacterial cultures (5 mL) grown to the late log phase were harvested by centrifugation. The cell pellet was resuspended in 5 mL 0.85% NaCl, aliquoted , and incubated with 4× MIC concentrations of the selected polymers for 4 h at 37 °C. The live cell control consisted of an aliquot incubated with only 0.85% NaCl. After incubation, all samples were washed with 0.85% NaCl twice. A Live/Dead BacLight Bacterial Viability Kit (Invitrogen) was used to check cell viability. The assay was performed according to the kit instructions. Component A (200 µL of a 3.34 mM solution in DMSO) was mixed in equal proportions with Component B (200 µL of a 20 mM solution in DMSO) and 3 μL of this combined dye mixture was added to 1 mL of all bacterial samples before incubation for 30 min in the dark. Samples were analyzed using an AxioVert 200M inverted fluorescent microscope to analyze for green  and red  fluorescence.Outer membrane permeabilization assay: The method for the determination of the outer membrane depolarization assay was adapted from the literature [E. coli were grown to 1.0 OD. Cells were harvested by centrifugation, washed, and resuspended to 0.1 OD in 5 mM HEPES with 5 mM glucose at pH 7.2. Then, 10 µL of NPN stock was added to each 1 mL of bacteria to give a concentration of NPN of 30 µM. Next, 200 µL of bacterial suspension was added to each well of a 96-well plate as five replicates at 37 °C, followed by analysis on a fluorescent microplate spectrometer (BioTek Synergy HT) over 30 min (every 1 min) (λex 350 nm λem 429 nm). Then, 10 µL of polymer solution (4× MIC) was added to each well. Moreover, 1% v/v Triton X-100 was used as a positive control, while a bacterial suspension without polymer was used as a negative control. The increase in the fluorescence of NPN was monitored at 5-min intervals and the fluorescence values after 30 min plotted. All experiments were repeated three times independently.terature . First, Cytoplasmic depolarization assay: The method for the determination of the inner membrane depolarization assay was adapted from the literature [B. subtilis was cultured in media overnight and centrifuged (3000× g—5 min) to give a pellet. The pellet was washed with PBS and resuspended to a 0D600 of 1.0 with PBS containing 10% (v/v) broth. The bacteria were incubated with 3 µM calcein-AM for 1 h at 37 °C. The dye-loaded cells were collected by centrifugation (3000× g—5 min) and suspended to 0.1 OD in PBS. Then, 200 µL of bacterial suspension was added to sterile black-wall 96-well plates (three replicates) and monitored for 30 min  on a fluorescence plate reader. Then, 10 µL of polymer solution (4× MIC) was added to each well. Bacteria without polymers were used as a negative control, while 1% v/v Triton X-100 was used as a positive control. All experiments were repeated three times independently.terature . A stockM. luteus) and Gram-negative (E. coli)) of bacteria were incubated with 0.85% NaCl (control) and the polymers PDAMAC-mid (20 kDa), PVMBT-high (40 kDa), and PMETACL-high (40 kDa)  for two hours. After pelleting, the bacteria were fixed in a solution of 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) for 2 h and washed (3 × 10 min changes of 0.1 M sodium cacodylate buffer). Samples were then post-fixed in 1% osmium tetroxide (in 0.1 M sodium cacodylate buffer) for 45 min, before further 3 × 10 min washes were performed in 0.1 M sodium cacodylate buffer. Dehydration in graded concentrations of acetone  was followed by critical point drying using liquid carbon dioxide. After mounting on aluminum stubs with carbon tabs attached, the specimens were sputter-coated with 20 nm thickness of gold–palladium and viewed using a Hitachi S-4700 scanning electron microscope.Aliquots . HeLa cells were used at passage numbers below 15 and tested for mycoplasma contamination regularly. For cytotoxicity tests, cells were seeded in a 96-well plate at the density of 1 × 104 cells per well and cultured in a 100 μL of growth medium. After 24 h, the culture medium was replaced with polymer solutions (50 µL) prepared in growth media at increasing concentrations. The cells were further incubated for 24 h under the same conditions. After the incubation of cells with polymers containing media, all the solutions were carefully removed from the wells and 100 µL MTT solution (5 mg of MTT dissolved in 1 mL PBS and 9 mL serum-free DMEM without phenol-red) was added to each well in a light-protected environment. After 4 h, unreacted dye was removed by aspiration. Cells were carefully washed with PBS twice. Then, 100 μL of solubilizing solution (a mixture of 2-propanol and dimethyl sulfoxide in a 1:1 volume ratio) was added to each well to dissolve the formed formazan crystals. The solution was shaken for at least 30 min to ensure the dissolution of all crystals before measuring the absorbance at 570 nm on a BioTek Synergy H1 plate reader. Cells treated with complete medium only were taken as a positive control (100% viability) and cells treated with DMSO (100%) were taken as a negative control. All tests were repeated three times. The relative cell viability (%) with respect to the control was determined using the formula below:In vitro cytotoxicity tests were carried out using an MTT assay on HeLa cells, based on a previously published protocol, with some modifications . Cells wThe test was applied in the range of 1.25 µM to 1280 µM for the polymers .v/v) in PBS. Different concentrations of PDAMAC-mid (20 kDa), PVMBT-high (40 kDa), and PMETACL-high (40 kDa)  were prepared, followed by the addition of the 5% RBC suspension (150 µL). PBS buffer was used as a negative control, and Triton X-100 (1% v/v in PBS) was used as a positive hemolysis control. Tubes were incubated at 37 °C for 1 h with 300 rpm shaking in an incubator. Samples were then centrifuged , and 100 μL aliquots of supernatants were transferred into a 96-well microplate, where absorbance values were read at 405 nm using a plate reader. The hemolysis percentage was calculated using the absorbance values and the formula below:polymer is the absorbance of the polymer-treated cells’ supernatant, Anegative is the absorbance of the negative control (1% v/v Triton X-100-treated cells’ supernatant), and Apositive is the absorbance of the positive control (PBS-treated cells’ supernatant). All experiments were performed as independent triplicates, each consisting of triplicates.Assays were carried out following previously published protocols . Sheep rB. subtilis, M. luteus) and Gram-negative  bacteria, with MIC values as low as 0.9 µM. The antimicrobial action mechanisms of the polymers were determined using dye permeabilization assays and visualization by SEM, which showed the formation of “holes” on the bacteria and a highly distorted cell envelope. Cytotoxic evaluations of the polymers were carried out using MTT and hemolysis assays, and the best polymers showed great selectivity towards bacteria over eukaryotic cells. Overall, the polymer PMETACL, in its higher-molecular-weight form, showed strong inhibition of bacterial growth (MIC = 0.9–1.8 µM (16–32 µg/mL)), with a bactericidal mode of action against both Gram-negative and Gram-positive bacteria, and it also showed low mammalian cell toxicity, making it a potential candidate for biomedical applications.In this study, five quaternary homopolymers were screened on Gram-negative and Gram-positive bacteria to detect the most active antimicrobial polymer. Selected polymers  were synthesized with varying molecular weights  and with the higher-molecular-weight polymers showing (on a mole/mole bases) higher activity . PVMBT (40 kDa) and PMETACL (40 kDa) and PDADMACs (20 kDa) showed good growth inhibition against both Gram-positive (