Blast-induced traumatic brain injury (bTBI) is a leading cause of morbidity in soldiers on the battlefield and in training sites with long-term neurological and psychological pathologies. Previous studies from our laboratory demonstrated activation of oxidative stress pathways after blast injury, but their distribution among different brain regions and their impact on the pathogenesis of bTBI have not been explored. The present study examined the protein expression of two isoforms: nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 and 2 , corresponding superoxide production, a downstream event of NOX activation, and the extent of lipid peroxidation adducts of 4-hydroxynonenal (4HNE) to a range of proteins. Brain injury was evaluated 4 h after the shock-wave exposure, and immunofluorescence signal quantification was performed in different brain regions. Expression of NOX isoforms displayed a differential increase in various brain regions: in hippocampus and thalamus, there was the highest increase of NOX1, whereas in the frontal cortex, there was the highest increase of NOX2 expression. Cell-specific analysis of changes in NOX expression with respect to corresponding controls revealed that blast resulted in a higher increase of NOX1 and NOX 2 levels in neurons compared with astrocytes and microglia. Blast exposure also resulted in increased superoxide levels in different brain regions, and such changes were reflected in 4HNE protein adduct formation. Collectively, this study demonstrates that primary blast TBI induces upregulation of NADPH oxidase isoforms in different regions of the brain parenchyma and that neurons appear to be at higher risk for oxidative damage compared with other neural cells. TBI causes approximately 1.5 million deaths and hospitalizations annually in the United States.1–3 Blast-induced TBI (bTBI) is the most prevalent form of brain injury in soldiers in combat zones because of the widespread use of high explosives in the war zones, and an increasing number of cases has also been reported in civilian populations with the use of improvised explosive devices by insurgents.5T7 The main inducers of oxidative stress are reactive oxygen species (ROS), which include superoxide (O2⋅−), hydroxyl radical (HO⋅), and hydrogen peroxide (H2O2).9 ROS are normally produced in several metabolic reactions, including redox-reactions (oxidation/reduction), oxidative phosphorylation, and in a normal process of electron transport chain reactions. There are a number of enzymes that produce free radicals during their catalytic reactions, which include the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family, cytochrome P450 (CYP450), cyclooxygenase (COX), lipoxygenase (LOX), and xanthine oxidase.Among many pathological factors associated with either primary mechanical injury or secondary biochemical cascades, oxidative stress has been shown to play a major role in various models of TBI.2•−). NOX is comprised of subunits that are both plasma membrane-bound  and cytoplasmic , which spans across the lipid bilayers.11 A number of NOX isoforms were identfied in the brain, which include NOX1, NOX2, and NOX4, and their cellular distribution is highly dependent on the cell type. Neurons express both NOX1 and NOX2, microglia are enriched with NOX2, while only small amounts of NOX isoforms were identified in astrocytes.12The NADPH oxidase (NOX) is a multi-subunit enzyme that catalyzes the reduction of molecular oxygen and oxidation of NADPH to generate superoxide radicals  and their cellular distribution and changes in rats exposed to moderate blast TBI. Levels of superoxide and formation of protein adducts of 4-hydroxynonenal (4HNE) were also determined. Based on the evidence that blast overpressure waves travel through the whole brain, we hypothesize that blast-induced NOX-related oxidative stress will be diffuse across the brain. Moreover, we hypothesize that different neural cell types have differential susceptibility to the oxidative stress-inducing effect of the primary blast.Guide for the Care and Use of Laboratory Animals and were approved by Rutgers University Institutional Animal Care and Use Committee before experiments. Rats were divided into two groups .Adult 10-week-old male Sprague-Dawley (Charles River Laboratories) rats weighing 320–360 g were used in all the studies. The animals were housed with free access to food and water in a 12-h dark-light cycle at 22°C. All procedures followed the guidelines established in the A total number of 24 rats was used in this study as follows: immunoblotting (three controls and three blast injured); immunofluorescence studies (four controls and four blast-injured); superoxide production studies (five controls and five blast-injured). For immunofluorescence studies, each brain tissue was processed to obtain several sections (>10) from frontal cortex, striatum, hippocampus, thalamus, and cerebellum. Each of those sections were used for identification of NOX1 and NOX2 isoforms in neurons, astrocytes, and microglia by double immunofluorescence analysis. Similarly, for superoxide production, several sections from different brain regions of animals (five control and five blast-injured) were used for regional analyses.19 The primary shockwave generated in this shock tube has been validated against the pressure-time profiles measured experimentally in the live-fire explosion experiments10 and against theoretical pressure-time profiles associated with the detonation of C4 explosive (see Kuriakose and colleagues20 for details).Rats were exposed to a single blast wave at the Center of Injury Biomechanics, Materials and Medicine  in the 9-inch square cross section shock tube as described previously.1819 Sham control rats received anesthesia and noise exposure but without blast exposure—i.e., anesthetized animals were placed next to the shock tube, and then a single blast was fired.All rats were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) at 10:1 ratio administered via intraperitoneal injection. Rats underwent a single exposure to 180 ± 5 kPa peak overpressure  and euthanized 4 h post-TBI. All rats were mounted in the middle of the shock tube  in a prone position—i.e. were strapped securely to the aluminum plate using a cotton cloth wrapped around the body . For immunoblot analysis, rats were anesthetized and transcardially perfused with phosphate buffered saline  whereas for immunofluorescence studies, rats were first perfused with PBS followed by 4% paraformaldehyde (PFA); brains were then isolated and cryoprotected in 30% sucrose.g at 4°C. The protein concentration in the samples was estimated by bicinchoninic acid (BCA) method .Before evaluating spatial resolution of NOX1 and NOX2 protein changes in different brain regions by immunofluorescence, we first examined their levels in cerebral hemisphere and cerebellum by immunoblot. After perfusion with PBS, brains were excised from the cranial vaults, the whole left hemisphere and cerebellum were separately homogenized in ice-cold conditions using CellLytic-M (Sigma) using sonicator with probe amplitude set to 45%. Samples were then centrifuged at 14,000 × Subsequently, 10–20 μg of protein per lane was loaded into 4–15% SDS-PAGE gradient gels (Bio Rad). Proteins separated according to their molecular size were then transferred onto polyvinylidene difluoride (PVDF) membranes using Turbo Protein Transfer instrument (Bio Rad Laboratories) according to manufacturer instructions. Membranes were blocked with 5% milk dissolved in Tris-Buffered saline containing 0.1% Tween-20 (TBS-T) and incubated overnight at 4°C with NOX 1 antibody (Sigma-Aldrich) or NOX2 antibody  or 4HNE  at a dilution of 1:1000. Bands were visualized using Western Pico Chemiluminescence Substrate (Thermo Scientific) on Chemi Doc Imaging System (Bio Rad Laboratories). For densitometric quantitation of Western blots, images were digitized using a BioRad GS800 calibrated densitometer, and analyzed with BioRad Quantity One software.To evaluate the spatial changes of NOX1 and NOX2 proteins in different brain regions, as well as to identify cell-specific changes in discrete brain regions, we performed double-immunofluorescence studies of two isoforms of NOX with NeuN, glial fibrillary acidic protein (GFAP) and Iba1, markers of neurons, astrocytes, and microglia, respectively, in frontal cortex, striatum, hippocampus, thalamus, and cerebellum. Briefly, 4 h post-injury, both sham and TBI animals were transcardially perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA). After perfusion, the brains were removed from cranial vaults and incubated in 4% PFA for an additional 48 h and cryoprotected by immersing in 30% sucrose. Brains were then dissected into 2 mm thick sections using rat brain slicer (Kent Scientific Corp.) and embedded in OCT  media and frozen quickly in isopentane cooled to liquid nitrogen temperature. Frozen sections were stored at −80°C until ready for sectioning.Brain sections (20 μm thick) were prepared from the frozen tissue blocks, using Leica CM3050 cryostat, and immunofluorescence was performed. Briefly, tissue sections mounted on glass slides prepared from four individual animals in each group were washed with 10 mM PBS, fixed in ice-cold methanol (100%) solution for 10 min at −20°C. The tissue sections were blocked with 10% donkey serum at room temperature for 1 h in PBS containing 0.03% Triton X-100. Fixed tissues were incubated overnight at 4°C with respective primary antibodies to NOX1, NOX2, GFAP, NeuN, and Iba1. The details on the source, clonality, and the dilution of each antibody used are provided in Double immunofluorescence was performed using donkey-antirabbit Alexafluor 594 for NOX1 or NOX2 and donkey-antimouse Alexafluor 488 for GFAP, donkey-antirabbit Alexafluor 488 for NeuN, and donkey-antigoat Alexafluor 488 for Iba1. The specificity of each antibody staining was validated by excluding each primary antibody (negative controls) and visualized for any nonspecific fluorescence. The primary antibody specificity, however, was not validated independently by blocking the binding to tissue with the corresponding antigen. Slides containing different brain regions were digitized (20x magnification) using Leica Aperio Versa 200 fluorescent microscope and slide scanner. Fluorescence intensities in each region were quantitated using AreaQuant software (Leica Biosystems) and expressed as average fluorescence intensity/unit area.2.-) levels in different brain regions were measured using dihydroethidium (DHE) following the method of Kim and coworkers.21 Briefly, control and blast-induced animals  were injected with 5 mg/kg DHE , dissolved in dimethylsulfoxide (DMSO) intraperitoneally, and 4 h after blast, animals were transcardially perfused first with PBS followed by 4% PFA, brains excised, and 50 μm thin sections of different brain regions were prepared using Leica VT 1000S vibratome and mounted. DHE immunofluorescence in each region was visualized by digitizing the images using Leica Aperio Versa 200 slide scanner. Fluorescent intensities in each region were quantitated using AreaQuant software (Leica Biosystems) and expressed as average fluorescence intensity/unit area.Superoxide , red: 594 nm (NOX1), and green: 488 nm . We then manually outlined the regions of interest in different brain structures, and the fluorescence intensities in each brain region were quantitated using FLAreaQuantV1 algorithm (Leica Biosystems) and expressed as average fluorescence intensity/unit stained area.For each channel, we set a minimum intensity threshold value using control sections as reference that will exclude any background fluorescence caused by nonspecific binding of fluorescent secondary antibody, and the same threshold values were used to quantify both control and experimental groups. A maximum intensity threshold was also set to remove any oversaturation from excess fluorescent dye. The algorithm outputs the area of positive staining for each brain region, the average intensity of each channel, and intensity profile of each protein.post hoc test (Bonferroni) to determine individual group differences. Differences between means were assessed at the probability level of p ≤ 0.05, 0.01, and 0.001. GraphPad Prism 6.0 software was used in all analyses and preparation of plots.Data are presented as mean ± standard error of the mean (SEM). Between-group comparisons were made by one-way analysis of variance (ANOVA) with a 18 This study evaluated further the effect of moderate blast (180 kPa peak overpressure) on the early evolution of the protein expression of NOX isoforms (NOX1 and NOX2). Immunoblot analysis of NOX1 and NOX2 in the whole cerebral hemisphere showed a significant increase  (p < 0.05)  . To asse < 0.05) .We next examined regional variations in NOX1 and NOX2 protein levels in the following brain structures: frontal cortex, striatum, hippocampus, thalamus, and cerebellum. The rationale in examining these regions is as follows: (a) NOX1 and NOX 2 isoforms are ubiquitously expressed in all brain regions, (b) the effect of shockwave propagation over the entire brain is not known, and (c) to use NOX1 and NOX2 as markers to evaluate whether there exists any selective vulnerability of various brain structures, a phenomenon that has not been investigated previously.Under primary blast loading conditions, the pathological changes were found throughout the brain as indicated by changes in the fluorescent intensities of NOX1 and NOX 2 in different brain regions. Interestingly. various regions displayed a differential response. Thus, NOX1 levels in the frontal cortex showed a 49% increase; hippocampus showed the highest degree of increase (107%) followed by thalamus displaying a 90% increase . Total NThe regional variations in the levels of NOX2 are slightly different from that of NOX1. Total NOX2 levels were highest in the frontal cortex (>two-fold) followed by the striatum and hippocampus that showed the lowest increase . NOX isoTo evaluate cellular vulnerability to oxidative damage resulting from primary blast, changes in NOX1 expression patterns in astrocytes, neurons, and microglia were determined by double immunofluorescence staining. In addition, simultaneous quantification of NOX immunofluorescence using cell-specific markers will provide an insight into changes in its expression in different neural cells including astrocytes, neurons, and microglia.Cell-specific analysis of increase in NOX1 and NOX2 levels after bTBI compared with their baseline expression (controls) indicate that neurons display a higher increase compared with astrocytes and microglia. In addition, NOX1 and NOX2 increases were more pronounced in neurons in the hippocampus and thalamus, respectively, compared with the frontal cortex. These conclusions were deduced based on the double immunofluorescence analysis showing co-localization of NOX1 with NeuN (a marker protein for neurons), with GFAP (a marker for astrocytes) and with Iba1 (a marker for microglia).  and 5.23 Similar to NOX1, elevated levels of NOX2 expression were also found to be mainly co-localized with neurons  but also in the parallel direction to the wave propagation from the pre-frontal cortex to the cerebellum. The total tissue levels of NOX1 in the cerebellum not only increased, but this increase was highest in neurons . Such hi neurons .24 Because the present study showed increased protein levels of NOX isoforms, we then sought to examine whether increased NOX protein after blast has a functional significance. Accordingly, in vivo levels of superoxide in the frontal cortex, hippocampus, thalamus, and cerebellum were measured using DHE. Hippocampus displayed a robust level of the increase in superoxide  followed by thalamus and frontal cortex. It is noteworthy that the extent of the rise in superoxide production in the hippocampus and thalamus .31Several reports indicate the TBI resulting from different etiologies, including primary blast and blunt injuries, display oxidative stress during the evolution of its symptoms.32–35 Lipid peroxidation is a process under which oxidants such as free radicals attack various lipids containing carbon double bonds, and the resulting aldehyde products such as 4HNE generated by the lipid peroxidation directly modify amino acid structures in proteins and form adducts.36One of the major downstream effects of oxidative stress in many neurological disorders and TBI is the formation and accumulation of lipid peroxidation products.38 Because the present study clearly showed an increase in NOX proteins and increased levels of superoxide after bTBI, we sought to examine the levels of 4HNE adducts in discrete brain regions in rats subjected to moderate blast . Immunoblot analysis of 4HNE products identified two major bands corresponding to molecular weights of 70 and 100 kDa, which showed a trend toward an increase in cerebral hemispheres, hippocampus, thalamus, and cerebellum; however, such changes were not significant  does the blast TBI affect different brain regions equally assuming homogeneous spatial distribution of biomechanical loading, and (2) does the extent of the damage vary between different brain regions containing disparate ratios of neural cells ? Hence, we focused on the role of two different forms of superoxide producing enzymes and their downstream oxidative damage markers to understand both the spatial and cellular effects.39–41 Among many causal factors implicated in the pathophysiology of bTBI, oxidative stress represents an important early pathological outcome resulting from either the activation of free radical producing enzymes or downregulation of antioxidant defense mechanisms.42The bTBI has an unique pattern of biomechanical phenotype in that the external forces are dictated by the size and shape of the interacting body—in this instance, skull and brain structures. Further, the magnitude of forces generated depends on the contact area and interaction time that in turn determine the overall biomechanical loads at the tissue level. This loading triggers secondary biochemical cascades depending on the local tissue content. The cascade of secondary events usually develops over a period of hours to days that ultimately lead to additional neuropathological sequela.12 Studies reported increased activation of different isoforms of NOX in various forms of TBI. Accordingly, in a mouse model of controlled cortical impact (CCI), increased NOX2 expression was observed at 24–48 h after injury43 whereas Byrnes and associates44 found a delayed increase in NOX2 activity one month post-injury. In a rat model of CCI, however, increased NOX activity was observed as early as 1 h post-injury.44 Increased NOX expression was also found in different animal models of fluid percussion injury.46 In addition, apocynin, an inhibitor of NOX activation, showed protective effects on various models of blunt TBI.46 Together these studies highlight the critical role of NOX in the pathology of TBI.NOX is a superoxide producing enzyme and different isoforms of NOX, including NOX1, NOX2, and NOX4 have been identified in the brain.47–49 This could in part be because of the lack of a true understanding the role of biomechanical loading in blast injury from limited availability of a field validated shock tube that simulates battlefield injuries. In the present study, we therefore characterized the spatial resolution of two isoforms of NOX (NOX1 and NOX2) in various brain regions and their localization in neurons, astrocytes, and microglia 4 h after blast injury in a field validated shock tube.20While studies have established the importance of NOX in the pathophysiology of blunt TBI, there are limited studies of NOX in bTBI.18 Further, using immunofluorescence analysis, the present study identified regional variations in the expression pattern of NOX 1 in that the highest increase is found in the hippocampus followed by the thalamus. Interestingly, the frontal cortex showed higher expression in NOX2 compared with that in the hippocampus and thalamus. Other fine brain structures, including motor cortex, amygdala, and hypothalamus, also showed a significant increase in both the isoforms of NOX with the exception that NOX2 levels were significantly lower in a motor cortex . Studies have shown that all neural cells express different isoforms of NOX, including NOX1, NOX2, and NOX4.53–55 While the reason for apparent abundance of NOX2 in neurons in our study is not known, studies report that NOX2 upregulation in hippocampal pyramidal neurons drives neuropathology associated with psychosocial stress in rats.57 Further, studies also report that cellular injury to neurons after stroke resulted in an early upregulation of NOX2 in neurons (3–6 h after injury) whereas NOX2 was upregulated in microglia at later time points (72 h post-injury).59 Such temporal difference in the expression pattern of NOX2 between neurons and microglia is reasonable because neurons may be far more vulnerable acutely in the evolution of the injury process whereas microglial activation could be a relatively late phenomenon.Our results are in slight contrast to other reports wherein NOX2 is abundantly expressed in microglia.60–63 Increased superoxide production has been shown in different models of TBI.64–67 The present study observed a striking increase in superoxide production in the hippocampus, thalamus, and frontal cortex, areas where corresponding increases in NOX levels were found. Moreover, our studies showing a complete absence of O2•− in animals treated with apocynin (an inhibitor of NOX activation) before the exposure to blast injury reinforces the finding that the increase in O2•− is indeed mediated by NOX activation. Our results are also in agreement with previous studies showing protective effects of apocynin in attenuating oxidative damage in different models of TBI.69Superoxide is a major free radical produced in the brain by a variety of reactions, including disturbances in mitochondrial oxidative phosphorylation, increased production of arachidonic acid as a consequence of activation of phosphilipase A2 (PLA2), activation of xanthine oxidase as well as by the activation of NOX.70 It is therefore likely that lipid peroxidation reaction and subsequent 4HNE formation may not have achieved a threshold as to show significant changes at 4 h after blast because reactive oxygen species other than superoxide  may not have been increased in the early phase of blast injury.While in the present study, the superoxide levels were found to be strikingly increased in different brain regions, the levels of 4HNE adducts showed a trend toward the increase in two proteins with average molecular weights of 70 and 100 kDa, but did not show statistically significant changes. The reason behind the lack of significant changes is not known. The 4HNE products, however, can be formed via peroxidation of unsaturated fatty acids not only by superoxide radical but also by a variety of other reactive oxygen species, including hydroxyl, peroxyl as well as a varety of cyclic compounds.71Despite an observed increase in factors conducive to oxidative damage in neurons, in the present study, we did not observe neurodegeneration nor apoptotic cell death as investigated by fluorescent staining for Fluorojade C and cleaved caspase-3 4 h after blast injury (data not shown). Perhaps a 4 h window of blast injury may be too early to detect neuronal death. It is also likely that moderate blast may not cause neuronal death because of the diffuse nature of the shockwave unlike blunt injuries where neuronal death was observed close to the epicenter in the acute phase of injury.72 In the present study, our observation of the highest increase in NOX levels in neurons in the hippocampus and cerebellum after blast suggests that neuron rich regions may be at higher risk for oxidative damage than other brain regions. This is further supported by our observation of a striking increase in superoxide levels in the hippocampus after blast. Also, within the hippocampus, studies have shown that neurons in CA1 region are more vulnerable to superoxide than CA3 neurons.74The selective vulnerability of neurons to oxidative damage may be dictated not only by the propagation of primary shockwave, the so-called direct mechanical impact throughout the brain regions, but also in part influenced by the cellular density and cell types distribution in different brain regions. Recently, it has been shown that the cerebellum and hippocampus contain the highest density of neurons compared with a cerebral cortex although relative mass of the hippocampus and cerebellum is far lesser than cerebral cortex.75 In support of this tenet, it has been shown that the density (and perhaps subsequent nutrient supply) of brain capillaries was far less in the CA1 region of the hippocampus compared with the CA3 region, which likely places the CA1 region at higher risk for ischemia and hypoxia.76Moreover, it is interesting to note that NOX1 showed a greater increase in CA1 region compared with CA3 . Such hi77 and was validated via intracranial pressure measurements by our group (data not published). If this is true—i.e. the mechanical forces created by the shockwave are distributed uniformly, and considering the brain's highly anisotropic and heterogeneous organization—our data suggest the extent of the local damage would depend on the composition and microarchitecture of a specific brain region . The uniformity of the pressure field within the rat brain under blast loading conditions was demonstrated in a numerical modeln region .Our studies demonstrate that moderate blast injury causes an increase in NOX isoforms in various brain regions to a differential degree. Increased NOX isoforms correlated with a concomitant rise in superoxide levels in corresponding regions that show a higher increase in NOX expression indicating oxidative damage. In addition, protein oxidation product 4HNE showed a strong tendency to increase in different brain regions. Further, higher increase in NOX isoforms in neurons compared with other brain cells strongly suggests neurons are by far the most vulnerable to oxidative damage in the early evolution of injury pathology.


Escherichia coli strains grown in co-culture. The first of these was an activator strain that overexpressed the E. coli nitroreductase NfsA, and the second was a nitroreductase null recipient strain bearing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by genotoxic prodrug metabolites can only occur following their transfer from the activator to the recipient cells. This can be monitored both in fluorescence based microtitre plate assays and by flow-cytometry, enabling modelling of the abilities of diverse nitroaromatic prodrug metabolites to exit a Gram negative vector.Bacterial-directed enzyme-prodrug therapy (BDEPT) uses tumour-tropic bacteria armed with a genetically-encoded prodrug-converting enzyme to sensitise tumours to a systemically-administered prodrug. A strong bystander effect  is critical to maximise tumour cell killing and avoid bacterial self-sterilisation. To investigate the bystander effect in bacteria we developed a sensitive screen that utilised two  Specification TableValue of the ProtocolThe bystander effect  is essential to bacterial-directed enzyme-prodrug therapy (BDEPT), as failure of the prodrug to exit the activating bacterium will negate the therapy. We present here the first protocol to quantify the ability of activated prodrugs to exit a bacterial activator cell, based on co-culture of an activator strain expressing a prodrug converting enzyme with a recipient strain that can quantify drug-induced damage. Both flow cytometry and microplate methodologies are described.i.e., the bystander effect) is a critical aspect of enzyme-prodrug therapies [The ability of activated prodrug metabolites to transfer from activating cells to neighbouring cells •IPTG•Sterile culture vials/tubes for cell growth•Sterile 384-well microtitre plates•Shaking incubator for culture growth•Spectrophotometer for monitoring cell growth•Microplate photometer for monitoring cell growth and fluorescence•Flow cytometer for monitoring fluorescence of individual bacteria•Software appropriate for analysis of flow cytometry data•Activator strain - expresses the prodrug activating enzyme but does not report on presence of the active prodrug metabolite.•Recipient strain - does not express the prodrug activating enzyme but can report on receipt of the active prodrug metabolite.•Negative control strain - does not express the prodrug activating enzyme and does not report on presence of the active prodrug metabolite .•Positive control strain - expresses the prodrug converting enzyme and can report on receipt of the active prodrug metabolite (used to set fluorescence gates during flow cytometry and to optimise the prodrug concentration(s) used in the assay).E. coli nitroreductase NfsA, which exhibits high level activity with a broad range of nitroaromatic substrates [In our previous study  and hencbstrates .activator strain and recipient strain were the E. coli strains 7NT and SOS-R4 as previously described [E. coli strain W3110 with seven endogenous nitroreductase candidate genes  deleted to reduce background activation of nitroaromatic prodrugs together with deletion of the tolC transporter gene to restrict active efflux of unreduced prodrug. The 7NT activator strain was transformed with pUCX expressing a prodrug-converting nitroreductase, and additionally transformed with empty pCDFDuet plasmid to confer spectinomycin resistance. The recipient strain was SOS-R4 , further transformed with empty pUCX to confer ampicillin resistance. The negative control strain was 7NT transformed with empty pUCX; and the positive control strain was SOS-R4 transformed with pUCX expressing the prodrug-converting nitroreductase.Our To accurately model the bystander effect in bacteria, the prodrug concentration must be optimised to ensure acceptable growth inhibition of the positive control strain. We have previously found that a greater than 20 % decrease in culture turbidity in response to prodrug challenge, relative to an unchallenged control, can cause a reduction in output during SOS assays . To enabE. coli can be performed in 384 well microtitre plates (•Test condition: co-culture of activator strain (nitroreductase-expressing 7NT) and recipient strain (nitroreductase null SOS-R4) challenged with the prodrug.•Control condition: co-culture of negative control strain (nitroreductase null 7NT) and recipient strain (nitroreductase null SOS-R4) that are together challenged with the prodrug.Evaluation of cell-to-cell transfer of activated prodrug metabolites in e plates .•Test co1−1 ampicillin, 50 μg ml−1 spectinomycin and 0.2 % glucose (w/v)) were established in sterile 15 ml centrifuge tubes and incubated overnight for 16 h at 30 °C with shaking at 200 rpm.Overnight cultures of a nitroreductase-expressing 7NT activator strain, a nitroreductase null 7NT control strain, and the SOS-R4 recipient strain were inoculated in 3 mL aliquots of LBASG media (lysogeny broth supplemented with 100 μg ml2600 should exceed 2.5). Next, 0.5 mL of each overnight culture were used to inoculate distinct day cultures of 10 mL of LBASGI , which were then incubated at 30 °C, 200 rpm for 3 h. Prior to prodrug challenge all cultures were diluted using fresh LBASGI media to an OD600 of 0.8.The following morning, cultures were visually assessed to ensure each had achieved expected levels of turbidity  to be ascertained.dA protective barrier of 60 μL of sterile media was dispensed into the immediately surrounding wells to prevent uneven exposure to incubation conditions, which can influence the growth of cultures.eAfter dispensing all the day cultures into the microtitre plate, mix well by pipetting.f600 for all experimental wells on a microplate photometer.Record the ODg600 and GFP fluorescence (excitation 490 nm/emission 530 nm) should be recorded on a microplate photometer.Incubate the microtitre plate at 30 °C, 200 rpm for 3.5 h, after which the ODIn 384-well microtitre plates, 4–8 wells were filled with either 30 μL control media (LBASGI only) or 30 μL challenge media . See 4aDerive the mean fluorescence of the technical replicates for the Test condition and divide this by the mean fluorescence of the technical replicates for the corresponding Control condition.b600 for prodrug challenged versus unchallenged conditions) to ensure that the level of inhibition does not exceed 20 %.Assess growth inhibition  in LBASGI media without prodrug. Use to set lower limit of selection gate to remove background auto fluorescence.•Positive control strain (nitroreductase-expressing SOS-R4) expresses the prodrug converting enzyme. Use to set upper limit of selection gate to capture the highest expected fluorescence in co-cultures.Conditions to set fluorescence selection gates:1aMake sure to include the individually challenged negative and positive control strains to establish the lower and upper flow cytometer parameters respectively.Pool each set of technical replicates for each challenge condition from the microtitre plate into individual micro centrifuge tubes A.aMake s2Prepare 14 mL round-bottom polypropylene tubes (or tubes appropriate to the flow cytometer you have access to) by dispensing 1 mL of filter sterilized Phosphate Buffered Saline pH 7.4 (PBS buffer) per tube.3aSet to lowest flow rate available to more accurately assess fluorescence of the individual bacteria.biBegin sample collection on flow cytometeriiAdjust the FSC and SSC values until you can identify the bulk of the bacterial population in the real-time dot plot display.Pipette 25 μL of the pooled no-prodrug cultures for the negative control strain into one of the polypropylene tubes containing PBS and mix by vortexing for five seconds.ciPipette 25 μL of the pooled no-prodrug cultures for the negative control strain into one of the polypropylene tubes containing PBS and mix by vortexing for five seconds.iiE. coli auto-fluorescence. Set the selection gate immediately above this boundary.Begin sample collection and establish the level of background fluorescence owing to iiiSelect the prodrug conditions you are going to collect data for. Pipette 25 μL of the pooled culture sample for the positive control strain challenged with that prodrug into one of the polypropylene tubes containing PBS and mix by vortexing for five seconds.ivThe positive control strain is used to set the upper limit of the selection gate to capture the highest expected fluorescence in co-cultures.vAdjust the GFP parameters of the flow cytometer until the cells with the highest GFP fluorescence in the population are captured and displayed in the dot plot and histogram. An upper limit is achieved when no significant number of cells is captured beyond this GFP parameter.viFor each new prodrug tested, reset the upper limit of the selection gate to the upper-most GFP signal recorded for the positive control strain challenged with that prodrug.Set selection gate using histogramsdOn a Becton Dickinson FACSCanto II  our typical parameters were FSC 570, SSC 450 and GFP 555. All data was collected within 2.5 h of removal from the incubator for each biological replicate, using the lowest flow rate of approximately 12 μL of sample per minute (BD LSR II User’s Guide).Setting up flow cytometer4aFor the pooled technical replicates of each prodrug challenge condition, pipette 25 μL of the pooled culture sample into one of the polypropylene tubes containing PBS and mix by vortexing for five seconds. For each sample collect 30,000 fluorescent events within the selection gate using a low flow rate while recording all data. It is advisable to also monitor the proportion of events recorded outside the selection gates; if this proportion is substantial then it may be necessary to re-set the selection gate boundaries.bMonitor the geometric mean of the GFP fluorescence for all events within the selection gate.ciMonitor and record overlap in the selection gate for the technical replicates using overlay histograms B.Perform this sample collection on the flow cytometer on two more 25 μL aliquots of the pooled sample, to generate three technical flow cytometry replicates for each Test and Control condition.Collecting data for the Test and Control conditions5aiFlowing Software version 2.5.1 by Perttu Terho .iiFlowjo .First review the exported data from the flow cytometer and record the individual GFP geometric mean for each technical flow cytometry replicate using appropriate software. We suggest one of the following programs:biAverage the geometric mean recorded from the three technical replicates of the Test condition then divide this by the average geometric mean recorded from the three technical replicates of the Control condition. Repeat for each prodrug tested.iiValues significantly >1 indicate activation of fluorescence in the recipient cell population as a consequence of the bystander effect.To generate each biological replicate, calculate the fold difference in GFP geometric mean of the Test condition versus the Control condition, for the events recorded within the selection gates C.iAveragcWe typically performed at least three biological replicates, derived from independent overnight cultures, for each prodrug condition under investigation.Processing the flow cytometry dataIn previous work we have described our development of a GFP-SOS reporter strain to quantify activation of nitroaromatic prodrugs by an over-expressed bacterial nitroreductase  and our The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


To investigate treatment outcomes and associated characteristics of persons experiencing homelessness who received 12-weekly doses of directly observed isoniazid and rifapentine (3HP/DOT) treatment for latent TB infection (LTBI).Among homeless persons treated with 3HP/DOT during July 2011 –June 2015 in 11 U.S. TB programs, we conducted descriptive analyses of observational data, and identified associations between sociodemographic factors and treatment outcomes. Qualitative interviews were conducted to understand programmatic experiences.Of 393 persons experiencing homelessness , 301 (76.6%) completed treatment, 55 (14.0%) were lost to follow-up, 18 (4.6%) stopped because of an adverse event (AE), and 19 (4.8%) stopped after relocations or refusing treatment. Eighty-one (20.6%) had at least one AE. Persons aged ≥65 were more likely to discontinue treatment than persons aged 31–44 years. Programs reported difficulty in following up with persons experiencing homelessness because of relocations, mistrust, and alcohol or drug use.This study demonstrates the feasibility of administering the 3HP/DOT LTBI regimen to persons experiencing homelessness, a high-risk population. Mycobacterium tuberculosis without evidence of active TB disease, a condition known as latent tuberculosis infection (LTBI). Reactivation of LTBI, compared with rapid progression from recent infection, is estimated to account for greater than 80% of new TB cases in the United States. or interferon-gamma release assay [IGRA]) at the time of TB evaluation, became pregnant during LTBI treatment, or were human immunodeficiency virus (HIV)-infected and on antiretroviral medications. The defiProgram sites were designated as being in basic (two sites), standard (four sites) or comprehensive (five sites) tiers, depending on the detail of data collected. All sites collected the following patient information: demographics, weekly dose and symptom monitoring for adverse reactions to the regimen at each directly observed treatment (DOT) visit, baseline and monthly laboratory monitoring, and final disposition. Sites in the standard and comprehensive tiers collected additional information regarding medical or behavioral risk factors and concurrent medications during 3HP/DOT treatment.Quantitative data from 11 sites were entered into a Microsoft Access  database and analyzed by using SAS version 9.3 . Premature treatment discontinuation was defined as receipt of <11 doses of 3HP/DOT over 16 weeks. For descriptive statistics, we determined frequencies and percentages by patient characteristics. We also described frequencies and percentages of patients reporting adverse events (AEs) with and without treatment discontinuation. An AE was defined as any symptom reported since last receipt of a 3HP/DOT dose. Throughout treatment, patients were monitored for symptoms including those common to rifapentine and isoniazid using an identified symptom review checklist. At each P values of < .05 as statistically significant. We also examined the effect of factors associated with 3HP treatment discontinuation using two multivariable log-binomial models. Factors considered for initial inclusion in the models were chosen based on the statistically significant findings from our bivariate analyses and clinical judgement of our subject matter expert. For the first model, we looked at the effect of patient socio-demographic characteristics and treatment reason on 3HP treatment discontinuation (N = 393). The second model only included patients from sites that collected data on medical and behavioral risk factors (N = 329), and examined the effect of socio-demographic characteristics, treatment reason, medical and behavioral risk factors on 3HP treatment discontinuation (N = 329). In both multivariable models, associations between covariates and 3HP/DOT treatment discontinuation were considered significant at a level of P < .05.Among persons experiencing homelessness, we conducted bivariate log-binomial analyses to assess independent associations between patient characteristics and treatment discontinuation. We reported relative risk (RR) values with their 95% confidence intervals (CIs). We considered Qualitative data were collected through in-depth interviews with health care workers from a convenience sample of three program sites that treated a substantial proportion of study patients experiencing homelessness. The interviews were conducted to elicit responses about the programmatic experience of using 3HP/DOT to treat LTBI and to gain a more thorough understanding of quantitative results.e.g., 3HP/DOT implementation start date, cost implications and staffing requirements). The second theme focused on the process of 3HP/DOT implementation, with certain questions relating to use of incentives and DOT while administering 3HP/DOT as well as description of the 3HP/DOT implementation process. Finally, questions related to the 3HP/DOT implementation process elicited provider perceptions of 3HP/DOT by those involved in the cascade of care.  ; 308 78. Eighty-P = 0.04630). Contacts of persons with active TB were less likely to discontinue treatment   was most frequently reported as a preexisting medical condition, followed by mental health problems (10%), and diabetes (6%). Twenty-eight percent were current or past smokers; 15% reported alcohol use (defined as more than 2 drinks per day); and 11% were non-injection-drug users . Most fr .00020) . No deatIn the two final multivariable models, no factor was significantly associated with 3HP/DOT treatment discontinuation,—neither when controlling for the association of socio-demographic factors with 3HP/DOT treatment discontinuation nor when controlling for medical and behavioral factors.e.g., informational brochures, personnel, and mental health or addiction services) to improve outreach to this population. Staff at one site emphasized using clear communication language throughout the entire spectrum of the LTBI testing and treatment cascade. As recommended, 3HP/DOT was administered by DOT at all sites; one site preferred administering medications in the clinic setting, whereas others preferred administering them at their local shelters.Three sites  participated in qualitative interviews. All three sites primarily targeted LTBI testing of persons experiencing homelessness, although one site tested additional populations. Staff perceived 3HP/DOT implementation cost to be high compared with standard 9H treatment at all three sites; nonetheless, staff understood that treating LTBI was a worthy investment for prevention of future, more-costly TB cases. Program staff did not receive additional funding for 3HP/DOT implementation; one program manager had convinced providers serving their homeless population outside of the health department setting to seek Medicaid reimbursement for 3HP/DOT. Only one site had hired additional staff (a nurse practitioner) to assist. All sites agreed that dedicated staff time or additional staff were required for expansion of services to contacts at homeless shelters. Sites used multiple resources and tools  or transportation vouchers; one of these sites had a treatment completion rate of 98%. Site staff expressed personal beliefs that such incentives as bus tokens and gift cards were effective in increasing adherence when given to patients. Site staff did not make substantial changes to their protocols to accommodate treating persons experiencing homelessness.Two sites provided incentives to patients, including food  in administering and monitoring treatment.Programmatic challenges mentioned by clinic staff included staffing and time requirements for treatment by DOT, patients’ difficulty with 3HP/DOT pill burden (three 300-mg pills/dose for isoniazid and six 150-mg pills/dose for rifapentine), higher costs of 3HP/DOT relative to 9H, and difficulty in following up with persons experiencing homelessness. Frequently reported AEs included high blood pressure while on 3HP/DOT medication. Previous and current substance users also reported concerns with triggering feelings or psychological feelings of being intoxicated (“high”) during blood draws.All sites agreed that the short duration of 3HP/DOT held great promise for increased adherence and higher treatment completion rates needed for TB prevention among this population.Although multivariable analyses did not yield significant medical, sociodemographic, or behavioral factors associated with treatment discontinuation, results from this study demonstrated that 3HP, a regimen of 12 doses of once-weekly rifapentine and isoniazid with DOT, was successful as a LTBI treatment option among persons who are reported as experiencing homelessness. The US Preventive Services Task Force recommends screening for and treating LTBI among persons in this high-risk population. the treaAs elicited from the interview responses, factors contributing to the high completion were presumed to include short duration of the regimen, , 23 low e.g., unstable housing, food insecurity, and high prevalence of mental health conditions),[P < .001).[Past studies have documented that difficulty in treating LTBI among persons experiencing homelessness is principally a result of issues of instability (ditions),–29 and n < .001)., 30e.g., monetary incentives and education programs) improved adherence.[Our qualitative data collection efforts indicated that the relatively high treatment completion rate achieved with 3HP/DOT also may be attributed to the implementation process adopted by the programs. Two of the three interviewed programs (with treatment completion rates of 98% and 69%) administered treatment with incentives, ranging from food and transportation vouchers to gift cards. Past studies have demonstrated that targeted strategies among the homeless population  because these factors were based on self-reporting. Lastly, the convenience sampling method used in selecting patients for the project may limit the generalizability of findings to other sites.Given that this study was conducted under field conditions at programmatic sites, this paper includes certain limitations. First, the study was observational; self-selected sites implemented the project under their program’s routine practice, which was not standardized across sites. Given the observational design of this project, all persons experiencing homelessness and requiring LTBI treatment may have not been captured, leading to an underestimation of this cohort of patients. Possible limitations in interpreting completion rates might also exist because certain programs differed in instructions for stopping treatment as a result of an adverse event. Other limitations include underreporting of risk factors for low completion rates  for latent tuberculosis infection (LTBI) among homeless persons in U.S. programmatic settings.Purpose of projectThe project is intended to assess and analyze factors contributing to and limiting treatment completion among homeless persons, and to eventually disseminate project findings through publication in a scientific journal.The interview aims to elicit responses about your site’s programmatic experience surrounding the use of 3HP for treating homeless persons with LTBI. Other programs planning on using this regimen for treating homeless persons will benefit from the information gathered.Key informant interview questionsProgram QuestionsWhen did your program start using 3HP for LTBI treatment ?Was the use of 3HP targeted at a specific population(s); If so, which ones?Did your program initially plan on treating homeless patients at the start of 3HP implementation?What were the cost implications of treating homeless patients with 3HP?How were project activities funded? Did you have to seek outside funding to support use of 3HP in the homeless population? Did you obtain any in-kind support for use of this regimen?Did the 3HP program require more staffing to carry out protocol activities?What resources and tools did you find useful to communicate effectively with homeless patients about taking 3HP?What other resources and tools would be useful for clinics and health care providers on using 3HP with homeless patients?Process Questions9Why did you decide to initiate use of 3HP for LTBI with homeless patients?10How did you determine or define if patients were homeless?11How did you determine if patients had LTBI? Which diagnostic tests were used?12What were your eligibility (inclusion and exclusion) criteria for treating a homeless patient for LTBI with 3HP?13Was the inclusion and exclusion criteria for homeless different from other populations?14Can you describe the overall process of 3HP implementation to target LTBI treatment among homeless patients?15Was treatment administered via DOT (directly observed therapy) or SAT (self-administered therapy), or both?16If DOT was used, was 3HP administered in clinical settings? Can you describe the settings ? Was 3HP provided to patients in other settings, like shelters or meal programs?17Was food provided to patients prior to or immediately after their dose?18Were any incentives provided? Can you describe the types of incentives used?19Did some incentives work better than others? If so, please describe which ones worked better and why you think that?20How were side effects monitored and assessed? Was this done differently than how it is routinely done in the clinic?21Was there a need to change site protocol to accommodate the use of 3HP among persons experiencing homelessness?22Were there partners in the delivery of care, and what was their role in the implementation process?Perception Questions23What were the perceptions and expectations of using 3HP before your program started using it?24What do you find challenging about using 3HP in persons experiencing homelessness?25What were the positive experiences homeless patients expressed about taking 3HP?26What were the negative experiences homeless patients expressed about taking 3HP?27Was there a difference in the implementation of 3HP among homeless patients compared to stably-housed patients? If so, please describe.28Was there a difference in the implementation of 3HP in the homeless population compared to other regimens for treating LTBI?29What do you like best about using 3HP to treat homeless patients, in comparison to other treatment regimens?30What additional challenges do you think other health care providers or health facilities might face in adopting the use of 3HP in their health care setting?31Based on your experiences, do you feel that 3HP should be or should not be used in the homeless patient population? Please explain why.S1 Table(XLSX)Click here for additional data file.


While recent work has revealed how different inhibitory interneurons influence responses of cortical neurons to sensory stimuli, little is known about their distinct contributions to sensory perception. Here, we optogenetically activated different genetically defined interneurons  in visual cortex (V1) of mice working at threshold in a contrast increment detection task. The visual stimulus was paired with optogenetic stimulation to assess how enhancing V1 inhibitory neuron activity during visual processing altered task performance. PV or SST activation impaired, while VIP stimulation improved, contrast increment detection. The impairment produced by PV or SST activation persisted over several weeks of testing. In contrast, mice learned to reliably detect VIP activation in the absence of any natural visual stimulus. Thus, different inhibitory signals make distinct contributions to visual contrast perception. Inhibitory interneurons are diverse and influence sensory responses through multiple mechanisms. Currently there is little consensus on how different inhibitory signals affect sensory perception. Here, we investigated how genetically defined interneuron subclasses  influence visual contrast perception. Mice were trained to work at threshold in a visual contrast detection task and interneuron activity was enhanced using ChannelRhodopsin-2 (ChR2) on a subset of trials. Different interneuron classes influenced perception in distinct ways. PV and SST stimulation impaired, whereas VIP stimulation improved visual contrast perception. With training, mice learned to perceive VIP activation in the absence of natural visual stimulation. These data highlight how different inhibitory signals affect the transformation of sensory-evoked responses into percepts.In the visual system, inhibitory interneurons contribute to fundamental sensory computations like stimulus selectivity , responsTransgenic mice that provide access to PV, SST, or VIP neurons for optogenetic targeting have helped clarify how different sources of inhibition augment stimulus-evoked responses in sensory cortices. PV and SST neurons, which target somata and dendrites, respectively , suppresComparatively little is known about how different sources of inhibition influence sensory perception. Perturbing PV neurons has produced conflicting results: some studies show that PV stimulation improves perceptual performance , while oHere, we expressed the excitatory opsin Channelrhodopsin-2 ChR2;  in eitheAll animal procedures followed NIH guidelines and were approved by the Institutional Animal Care and Use Committee of the University of Chicago. Mouse lines were obtained from The Jackson Laboratory. Data come from PV-Cre mice . Using aseptic technique, a headpost was secured using adhesive cement  and a 3-mm craniotomy was made over the left cerebral hemisphere  to implant a glass window .Each mouse (three to five months old) was implanted with a headpost and cranial window to give stable optical access for photostimulation during behavior . AnimalsWe located V1 by measuring changes in the intrinsic autofluorescence signal using epifluorescence imaging . Autoflu11 viral particles; Penn Vector Core) 300 μm below the pial surface. The virus was injected at a rate of 40 nl/min through a glass capillary attached to a syringe (Hamilton). Following the injection, a new cranial window was sealed in place. Several weeks after injection, we localized the area of ChR2 expression using tdTomato fluorescence. We fit a Gaussian to ChR2 fluorescence to map the area of expression. The area of ChR2 expression was similar across the three genotypes . We attached an optical fiber  within 500 μm of the cranial window . We delivered 455-nm light from an LED (Thorlabs) through the fiber. Power calibrations were based on light at the entrance to the cannula. Optogenetic stimulation began no earlier than six weeks after injection. We prevented optogenetic stimuli from cueing the animal to respond by shielding the fiber implant with blackout fabric (Thorlabs) secured to the headpost using a custom mount.ChR2 injections were targeted to the monocular region of V1 based on each animal’s retinotopic map . For injection, mice were anesthetized  and the cranial window was removed using aseptic technique. We used a volume injection system (World Precision Instruments) to inject 200 nl of AAV9-Flex-ChR2-tdTomato (∼10http://mworks-project.org), MATLAB , and Python.Animals were water scheduled and trained to respond to changes in a visual display using a lever . AnimalsOptogenetic stimulation did not begin until animals worked reliably for hundreds of trials each day and contrast detection thresholds were stable across days. This typically required approximately two months of training. During optogenetic experiments, we activated ChR2-expressing inhibitory neurons on a randomly selected half of the trials by delivering light through the optical fiber positioned over V1. During preliminary sessions , the stimulation power was adjusted to produce an approximately two-fold change in detection threshold . Once selected, the stimulation power was fixed throughout testing. To align the opsin illumination with visually evoked spiking in V1, the onset of illumination was delayed by 35 ms relative to the appearance of the visual stimulus on the monitor.Mice were perfused with 10% pH neutral-buffered formalin (Millipore Sigma Inc.), after which the brain was removed and submerged in fixative for 24 h. The brain was subsequently rinsed with PBS, placed in a 30% sucrose PBS solution until it sank. Brains were sectioned at 40 μm on a freezing microtome, mounted and cover slipped. tdTomato expression was visualized with 561-nm excitation using a Caliber ID RS-G4 Confocal Microscope with a 10× objective .unstimulated, αstimulated), a common lapse rate (γ), and a common slope (β) such that:Detection thresholds were determined using trials in which the subject either responded correctly (hit) or failed to respond miss; . PerformF test for number of parameters). Sessions fit with five parameters were distributed across genotypes  and produced similar effects of stimulation on psychometric detection thresholds  with four parameters = 1.54; median threshold change with five parameters = 1.45; p = 0.35; Wilcoxon rank-sum test). Threshold confidence intervals were estimated using a bootstrap .A small fraction of sessions (19/129) were significantly better fit by the addition of a second slope parameter specific to ChR2 stimulation trials . For comparing models, performance data were not corrected for false alarms as was done previously so that we could better assess whether ChR2 stimulation produced a change in the probability that the animal would respond independent of the visual stimulus. All three models had the same number of parameters. Trials without ChR2 stimulation were fit with a threshold (α), lapse rate (γ), slope (β), and false alarm rate (δ) such that:To assess how activating different interneuron classes influenced task performance, the data were fit to three different models, which were compared using log-likelihood ratios, with confidence intervals estimated using a bootstrap procedure  that augmented the psychometric function differently depending on the model being tested. We first tested a contrast gain model in which ChR2 stimulation causes a multiplicative change in the contrast of the stimulus.We also tested a contrast addition model, in which ChR2 stimulation adds a fixed increment or decrement to the contrast of the stimulus.Where σ was negative, we constrained the fit such that the proportion correct was δ  whenever contrast + σ was negative, under the assumption that optogenetic stimulation should not impact spontaneous lever releases.Lastly, we tested a model in which ChR2 stimulation augments the probability of lever release (response probability) independent of the visual stimulus. This model takes different forms depending on whether the response probability is decreased:n = 17 sessions from two mice).In the case of ChR2 detection in the absence of visual stimuli (VIP-Cre mice), we fit a three-parameter psychometric function  after correcting for false alarms as above. False alarm rates in ChR2 detection experiments were comparable to experiments with both visual and optogenetic stimuli . Visual stimuli were presented at a visual field location corresponding to the V1 representation with strongest ChR2 expression B. We varp = 0.74; Kruskal–Wallis test).We began optogenetic stimulation experiments after contrast increment detection thresholds were stable. The optogenetic stimulus power was held constant throughout a session at a level chosen ensure that mice could still reliably report high contrasts. Such settings produced an ∼2× change in threshold and roughly corresponded to the relative proportions of each interneuron subtype . Powers were selected during one or two preliminary screening sessions before experimental data collection that were not included in the main analyses. We measured the effects of optogenetic stimulation by fitting Weibull functions to behavioral data collected with and without optogenetic stimulation . To isolp < 10−8; Wilcoxon signed-rank test; Optogenetic stimulation of PV neurons impaired contrast increment detection, resulting in rightward shift of psychometric functions A. Acrossp < 10−7; Wilcoxon signed-rank test; average ratio 2.2, SEM 0.1; range 1.1–5.1; Optogenetic stimulation of SST interneurons similarly impaired contrast increment detection, shifting psychometric functions to the right C. Acrossp < 10−7; Wilcoxon signed-rank test; In stark contrast to PV and SST perturbations, optogenetic stimulation of VIP neurons enhanced contrast increment detection, shifting psychometric functions to the left E. AcrossEnhancing the activity of cortical interneurons augments sensory-evoked spiking in pyramidal output cells . If our We tested these competing possibilities by fitting the behavioral performance from each session to all three models  and computing the log-likelihood ratio for pairs of models . For PV and SST mice, we found that the contrast gain model was superior to a model in which optogenetic stimulation reduced the response probability. For PV stimulation, the contrast gain model was favored over the lever release model in 91% (43/47) of sessions : PV or SST stimulation never improved performance. The slopes of lines fit to the example data from the PV and SST mice were not significantly different from zero (PV: t(14) = –1.78, p = 0.1; SST: t(16) = –0.77, p = 0.45; F test for linear regression), suggesting that the impairment produced by optogenetic stimulation of PV or SST neurons was stable across sessions. No PV or SST mouse showed a significant reduction in the threshold impairment during our experiments. Thus, mice did not learn to detect or discount PV or SST activation.To examine whether animals could detect V1 interneuron activation in the absence of a visual stimulus, we tracked optogenetically induced changes in threshold across daily sessions. p = 0.66; Wilcoxon rank sum test). We attribute the change to the mice directly detecting a percept created by the activation of VIP neurons , and using it to guide their behavioral responses. To examine this directly, we tested whether mice could be trained to respond to VIP neuron stimulation in the absence of any natural visual stimulus. By progressively lowering the contrast of the visual stimulus, animals were conditioned to respond 100-ms periods of VIP interneuron activation in isolation  in orientation, where sharpening of tuning curves should have little effect on discrimination. Subsequent work showed that optogenetic stimulation of PV neurons impaired orientation discrimination thresholds  and neuronal activity that could generate responses independently from the visual stimulus (response model). Given that none of the three models we tested for VIP mice was clearly superior, we cannot conclusively distinguish between these competing possibilities. Future studies will be needed to resolve the nature of VIP-mediated effects on visual perception.Given that optogenetic activation of V1 pyramidal neurons is perceptible  and VIP


FST genetic distances across genome-wide SNP panel, we detected diversifying selection signatures among these two subpopulations and indicated (among others) the significance of DGAT1 and FGF2 genes for milk production traits in these cattle. We also found that among genes being presumably under selection in terms of milk production, there are genes responsible, for example, for mammary gland development  and immune system response . The most important finding of this study is that the most pronounced genetic differences between the analyzed populations were associated with β-defensin genes  located within so-called bovine cluster D on BTA27. The β-defensins are expressed mainly in the mammary gland and are antimicrobial peptides against the Gram-negative and Gram-positive bacteria, viruses, and other unicellular parasites. This suggests that antimicrobial resistance of mammary gland is of high importance during selection towards increased milk production and that genes responsible for this process are selected together with increasing levels of productivity.Polish Red cattle is one of the few indigenous breeds of European red cattle which is characterized by several desired features, such as high disease resistance, good health, longevity, good fertility, and high nutritional value of milk. Currently, Polish Red cattle population is a subject of two independent breeding programs: (i) improvement program and (ii) genetic resources conservation program. The aim of the improvement program is the genetic progress in terms of milk production and body conformation traits, while the conservation program mainly focuses on protection of the genetic resources of Polish Red cattle and preservation of the existing, original gene pool. By the analysis of The online version of this article (10.1007/s13353-019-00484-0) contains supplementary material, which is available to authorized users. Polish Red cattle is one of the few indigenous breeds of European red cattle. It is characterized by several features typical for primitive populations, such as high disease resistance, good health, longevity, very good fertility, easy births, ease of calf rearing, and high biological value of milk. The Polish Red cattle is also characterized by a good adaptation to harsh environmental conditions, which is especially visible in the ability to limit the efficiency , as well as the relatively quick regeneration after condition loss. These features make the cattle of this breed well adapted to mountainous and submountainous living and production conditions  improvement program and (ii) genetic resources conservation program  and “improvement”  breeding programs. The animals were randomly selected and verified to be unrelated for at least two generations. The animals were coming from at least three different herds. All animal procedures were approved by the Local Animal Care Ethics Committee No. II in Kraków—permission number 1293/2016 in accordance with EU regulations. The genomic DNA was purified using Sherlock AX kit  and after quality control was genotyped with the use of BovineSNP50v2 BeadChip assay  according to the standard Infinium Ultra protocol. Only samples with call rate > 0.95 were retained for analysis. The initial markers set included 54,609 SNPs and were further filtered to remove markers without known chromosomal position or located on sex chromosomes. SNPs that had minor allele frequency (MAF) < 0.05, genotyping rate < 0.8 (in joint populations), and deviated from Hardy-Weinberg equilibrium with p < 0.0001 (in each population separately) were also removed. This filtering step allowed retaining for further analysis a common set of 43,165 SNPs with average inter-marker distance of 57.9 kb in UMD3.1 genome assembly.The study material comprised 60 samples of ear tissue collected from cows belonging to Polish Red breed and included in “conservation”  and haplotype block structure at the most divergently selected regions among the studied populations were analyzed using HaploView 4.2  based on individual genotypes and mean or weighted The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.FST distances of 0.0226 and 0.0242, respectively. The PCA showed, however, that both populations form clearly separated clusters of observations with visibly higher level of genetic variation observed in RE population  allowed for detection of 46 overlapped genes. The overrepresentation test performed for those genes showed single enriched GO category connected with defense response to bacterium (adjP = 0.02). The genes associated with this process belonged mainly to the β-defensins family . In the analyzed genes set, we also detected other immune system functioning–related genes, like CD53 and TRAT1 . These two selection signals were analyzed in details to identify their possible meaning and evaluate haplotype structure at these loci. The analysis of the divergently selected region on BTA3 showed that the detected selection signal colocalized with the large chromosomal region of strong LD (31.6–33.5 Mb) and encompassed large haplotype block with high-frequency haplotypes in both studied populations (> 0.5). The detailed analysis of haplotypes showed that in separate populations, the most common haplotypes were composed of alternative SNP variants. Additionally, only haplotypes found in RP breed showed some minor signs of recombination  including one non-coding small nuclear RNA.Two selection signals found in this study included, inter alia, the strongest detected signal on BTA3 (32.5–32.9 Mb) and the signal spanning the largest genomic region (1.3 Mb) among the signals with top 0.1% of The genomic region on BTA27 overlapping with the detected large and strong diversifying selection signal was also characterized by strong LD  and encompassed three haplotype blocks showing common variants with haplotype frequency above 0.5. The detailed analysis of haplotypes again showed signs of diversifying selection among the analyzed populations, represented by haplotype variation and divergent selection of separate variants Fig. . The sigIn this study, we attempt to identify diversifying selection signatures among two subpopulations of Polish Red cattle formed by animals subjected to conservation program (selected mainly towards primitive type and breed standard) and animals used for milk production . These two populations visibly diversified over time and the phenotypical differences between them mainly refer to milk yield traits as well as exterior features which were strongly shifted towards milk production type in the improved cattle Fig. .Fig. 5Twloci which were divergently selected since the implementation of the improvement program. The performed PCA showed that the genomes of both these populations bear signs of differentiation with fraction of individuals being still genetically close. RE population was, however, subdivided into two visible clusters due to the different level of historical crossbreeding present in the pedigrees of the analyzed animals. The fact that both subpopulations originate from the same native population created an opportunity to trace allele frequency differences among them with high probability that they result from application of selection directed to improvement of milk traits in commercial population. This allowed for identification of candidate genes affecting presumably both primitive traits found in conserved population (being lost due to the intensive selection)  confirms their common origin from local population  and FGF2 (Fibroblast Growth Factor 2) , which may be of high importance for the improved population and its milking abilities  controls the size and shape of mammary buds in mice  ablation results in defective mammary morphogenesis and regeneration  was shown to play a role in development and reorganization of the mammary gland  gene. This gene was previously shown to have effect on body size regulation in horses , spanning over 1.1 Mb (32.4–33.6 Mb) in both analyzed populations. Within the detected selection signal, we found only three genes, namely LRIF1, CD53, and KCNA3. These gene functions could be connected with both male fertility and immune system response. Exemplary, LRIF1 product represses the ligand-induced transcriptional activity of retinoic acid receptor alpha (RARA) and disruption of retinoid signaling was shown to impair mammalian spermatogenesis and fertility  affecting milk yield, fat yield, and protein yield in Holstein and other cattle populations , the largest genomic region with signs of divergent selection was identified on BTA27 (1.29 Mb). This signal overlapped with several genes coding proteins belonging mainly to the β-defensins family , and non-lactating cows  has been also presented, where higher concentration of LAP in the milk of cattle infected with Staphylococcus aureus, Streptococcus bovis, Streptococcus dysgalactiae, and E. coli was observed than in uninfected cows  polymorphisms were significantly associated with protein yield and fat or protein contents Genomic regions formed by FESM 2ST values. (XLSX 61 kb)Genes found within top 1% of window-averaged F