Effects of Soaking and Germination Treatments on the Nutritional, Anti-Nutritional, and Bioactive Characteristics of Adzuki Beans (Vigna angularis L.) and Lima Beans (Phaseolus lunatus L.).

Lima beans (Phaseolus lunatus) and adzuki beans (Vigna angularis) are some of the most nutritious underutilized pulses that are significant in being used as basic ingredients for the preparation of various food products. The present study aimed to determine the impact of soaking and germination on nutritional and bioactive components, in vitro protein digestibility, reducing power, metal chelating capacity, antioxidant activity, and anti-nutritional components of lima and adzuki beans. The findings showed that during the germination treatment, the in vitro protein digestibility of lima and adzuki beans increased by 14.75 and 10.98%, respectively. There was an increase in the antioxidant activity of lima beans by 33.48% and adzuki beans by 71.14% after 72 h of germination, respectively. The reducing power assay of lima and adzuki beans indicated an increase of 49.52 and 36.42%, respectively, during germination. Similarly, the flavonoid and metal chelating activity increased in lima and adzuki beans after 72 h of germination. In contrast, the anti-nutrients, such as phytic acid, tannin content, and trypsin inhibitor activity, decreased significantly p < 0.05 after 72 h of germination. These results are encouraging and allow for utilizing the flour obtained from the germinated beans in functional bakery products, which can contribute to eradicating protein deficiency among some population groups. At the same time, promoting soaking and germination of the beans as a way to enhance the nutritional quality and reduce anti-nutrients can contribute to the interest in these underutilized pulses. They could be seen as an additional tool to improve food security.

Genetic analysis of iron, zinc and grain yield in wheat-Aegilops derivatives using multi-locus GWAS.

BACKGROUND: Wheat is a major staple crop and helps to reduce worldwide micronutrient deficiency. Investigating the genetics that control the concentrations of iron (Fe) and zinc (Zn) in wheat is crucial. Hence, we undertook a comprehensive study aimed at elucidating the genomic regions linked to the contents of Fe and Zn in the grain. METHODS AND RESULTS: We performed the multi-locus genome-wide association (ML-GWAS) using a panel of 161 wheat-Aegilops substitution and addition lines to dissect the genomic regions controlling grain iron (GFeC), and grain zinc (GZnC) contents. The wheat panel was genotyped using 10,825 high-quality SNPs and phenotyped in three different environments (E1-E3) during 2017-2019. A total of 111 marker-trait associations (MTAs) (at p-value < 0.001) were detected that belong to all three sub-genomes of wheat. The highest number of MTAs were identified for GFeC (58), followed by GZnC (44) and yield (9). Further, six stable MTAs were identified for these three traits and also two pleiotropic MTAs were identified for GFeC and GZnC. A total of 1291 putative candidate genes (CGs) were also identified for all three traits. These CGs encode a diverse set of proteins, including heavy metal-associated (HMA), bZIP family protein, AP2/ERF, and protein previously associated with GFeC, GZnC, and grain yield. CONCLUSIONS: The significant MTAs and CGs pinpointed in this current study are poised to play a pivotal role in enhancing both the nutritional quality and yield of wheat, utilizing marker-assisted selection (MAS) techniques.

Consensus genomic regions associated with grain protein content in hexaploid and tetraploid wheat.

A meta-analysis of QTLs associated with grain protein content (GPC) was conducted in hexaploid and tetraploid wheat to identify robust and stable meta-QTLs (MQTLs). For this purpose, as many as 459 GPC-related QTLs retrieved from 48 linkage-based QTL mapping studies were projected onto the newly developed wheat consensus map. The analysis resulted in the prediction of 57 MQTLs and 7 QTL hotspots located on all wheat chromosomes (except chromosomes 1D and 4D) and the average confidence interval reduced 2.71-fold in the MQTLs and QTL hotspots compared to the initial QTLs. The physical regions occupied by the MQTLs ranged from 140 bp to 224.02 Mb with an average of 15.2 Mb, whereas the physical regions occupied by QTL hotspots ranged from 1.81 Mb to 36.03 Mb with a mean of 8.82 Mb. Nineteen MQTLs and two QTL hotspots were also found to be co-localized with 45 significant SNPs identified in 16 previously published genome-wide association studies in wheat. Candidate gene (CG) investigation within some selected MQTLs led to the identification of 705 gene models which also included 96 high-confidence CGs showing significant expressions in different grain-related tissues and having probable roles in GPC regulation. These significantly expressed CGs mainly involved the genes/gene families encoding for the following proteins: aminotransferases, early nodulin 93, glutamine synthetases, invertase/pectin methylesterase inhibitors, protein BIG GRAIN 1-like, cytochrome P450, glycosyl transferases, hexokinases, small GTPases, UDP-glucuronosyl/UDP-glucosyltransferases, and EamA, SANT/Myb, GNAT, thioredoxin, phytocyanin, and homeobox domains containing proteins. Further, eight genes including GPC-B1, Glu-B1-1b, Glu-1By9, TaBiP1, GSr, TaNAC019-A, TaNAC019-D, and bZIP-TF SPA already known to be associated with GPC were also detected within some of the MQTL regions confirming the efficacy of MQTLs predicted during the current study.

Microbial consortium with nitrogen fixing and mineral solubilizing attributes for growth of barley (Hordeum vulgare L.).

Bioinoculants provide better opportunity for ecological farming practices to improve the plant growth and enhanced crop productivity. Different types of bioinoculants containing single microbial culture and multiple microbial strains in single formulation could be used for agricultural sustainability. The different efficient microbial strain in single formulation as a consortium is an emerging trend in the present era. The present study deals with the isolation of nitrogen fixing, phosphorus and potassium solubilizing microbes from rhizospheric soil and root's internal tissues of different cereal/pseudocereal crops and their application as a microbial consortium for the growth of cereal crops. A total of 152 rhizospheric and endophytic bacteria were isolated and screened for the plant growth promoting (PGP) traits of nitrogen fixation, solubilization of phosphorus, and potassium. Among all the isolates, nine were found to fix nitrogen, fifteen and eleven exhibited phosphorus and potassium solubilization activity, respectively. Three selected efficient bacterial strains were identified using 16S rRNA gene sequencing as Erwinia sp. EU-B2SNL1 (N-fixer), Chryseobacterium arthrosphaerae EU-LWNA-37 (P-solubilizer), and Pseudomonas gessardii EU-MRK-19 (K-solubilizer). The inoculation of these three bacterial strains on barley crop as single inoculum and as microbial consortium enhanced the growth and physiological parameters including root/shoot length and biomass, chlorophyll, carotenoids, phenolics, flavonoids and soluble sugar content in comparison with untreated control. The microbial consortium was found to be more effective as compared to single inoculum. The microbial consortium of nitrogen fixing and mineral solubilizing microbes could be used as biofertilizer for plant growth and soil health.

Viral expression of constitutively active AKT3 induces CST axonal sprouting and regeneration, but also promotes seizures.

Increasing the intrinsic growth potential of neurons after injury has repeatedly been shown to promote some level of axonal regeneration in rodent models. One of the most studied pathways involves the activation of the PI3K/AKT/mTOR pathways, primarily by reducing the levels of PTEN, a negative regulator of PI3K. Likewise, activation of signal transducer and activator of transcription 3 (STAT3) has previously been shown to boost axonal regeneration and sprouting within the injured nervous system. Here, we examined the regeneration of the corticospinal tract (CST) after cortical expression of constitutively active (ca) Akt3 and STAT3, both separately and in combination. Overexpression of caAkt3 induced regeneration of CST axons past the injury site independent of caSTAT3 overexpression. STAT3 demonstrated improved axon sprouting compared to controls and contributed to a synergistic improvement in effects when combined with Akt3 but failed to promote axonal regeneration as an individual therapy. Despite showing impressive axonal regeneration, animals expressing Akt3 failed to show any functional improvement and deteriorated with time. During this period, we observed progressive Akt3 dose-dependent increase in behavioral seizures. Histology revealed increased phosphorylation of ribosomal S6 protein within the unilateral cortex, increased neuronal size, microglia activation and hemispheric enlargement (hemimegalencephaly).

Effect of wheat grain protein composition on end-use quality.

The quality of wheat products has been a new challenge next to wheat production which was achieved substantially during green revolution. The end-use quality of wheat is an essential factor for its commercial demand. The quality of wheat is largely based on the wheat storage proteins which extensively influences the dough properties. High molecular weight glutenin subunits (HMWGS), low molecular weight glutenin subunits (LMWGS) and gliadins significantly influence the end-use quality. Genomics and proteomics study of these gluten proteins of bread and durum wheat have explored new avenues for precise identification of the alleles and their role in end-use quality improvement. Secalin protein of Secale cereale encoded by Sec-1 loci and is associated with 1RS.1BL translocation has been known for deterioration of end-use quality. Chromosomal manipulations using various approaches have led to the development of new recombinant lines of wheat without secalin. Advanced techniques associated with assessment of end-use quality have integrated the knowledge of useful or deteriorating HMWGS/LMWGS alleles and their potential role in end-use quality. This review gives a comprehensive insight of different aspects of the end-use quality perspective for bread making in wheat along with some information on the immunological interference of gluten in celiac disease.

A Study of Depressive Symptoms in Doctors Working at COVID-19 Hospitals: An Online Survey.

OBJECTIVE: COVID19 outbreak has put a tremendous physical and mental burden on frontline doctors. A limited amount of literature is available in this area. The present study was done to assess the depressive symptoms and depression levels in doctors working at COVID-19 Hospitals. METHOD: This study was based on an online survey that was started on May 2020 and ended on 30th June, 2020. An online questionnaire which included details such as age, gender, and 21 items to assess depressive symptoms was sent through social media to doctors from various countries. Depressive symptoms were measured by Beck's Depression Inventory-II (BDI-II). Items of a total of 220 questionnaires were responded. Out of these, only 200 responses were analyzed using SPSS software. RESULTS: Out of 200 subjects, 110 (55%) had depressive symptoms and 90 (45%) had no symptoms. Males had more depressive symptoms (42.5%) than females (12.5%). Those who worked at COVID-19 centers 75 (37.5%) had higher depressive symptoms. The number of males working at COVID-19 centers was much higher (36%) than females (12%). Depressive symptoms were significantly higher in males than females (35.35±10.25 vs 16.90±7.76; p<0.0001). CONCLUSION: Doctors, especially males working at COVID-19 centers have higher depressive symptoms than their female colleagues.Multicentric studies with larger sample sizes are needed to study the impact of COVID-19 on frontline doctors.

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord.

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

Retrogradely Transportable Lentivirus Tracers for Mapping Spinal Cord Locomotor Circuits.

Retrograde tracing is a key facet of neuroanatomical studies involving long distance projection neurons. Previous groups have utilized a variety of tools ranging from classical chemical tracers to newer methods employing viruses for gene delivery. Here, we highlight the usage of a lentivirus that permits highly efficient retrograde transport (HiRet) from synaptic terminals within the cervical and lumbar enlargements of the spinal cord. By injecting HiRet, we can clearly identify supraspinal and propriospinal circuits innervating motor neuron pools relating to forelimb and hindlimb function. We observed robust labeling of propriospinal neurons, including high fidelity details of dendritic arbors and axon terminals seldom seen with chemical tracers. In addition, we examine changes in interneuronal circuits occurring after a thoracic contusion, highlighting populations that potentially contribute to spontaneous behavioral recovery in this lesion model. Our study demonstrates that the HiRet lentivirus is a unique tool for examining neuronal circuitry within the brain and spinal cord.

Prevalence and prognostic relevance of BrafV600E mutation in colorectal carcinomas from Kashmir (North India) valley.

Molecular studies have implicated mutant B-type Raf kinase (BRAFMut) of MAP-kinase signalling pathway in the pathogenesis of several cancers including colorectal cancer. Recently, the prognostic and therapeutic relevance of the most frequent BRAFV600E mutation also has been highlighted in colorectal carcinomas (CRC). Thus, the aim of this study was to investigate the prevalence of BRAFV600E mutation and to determine the correlation between this mutation and indicators of poor prognosis and outcome in patients with CRCs from Kashmir, North India. Here, we developed a highly sensitive technique, mutation allele-specific multiplex PCR (MASMP), for detection of BRAFV600E/BRAFc.1799T>A mutation, the results of which were confirmed by sequencing the product and compared to direct DNA sequencing. In total, BRAFV600E mutation status was analyzed in 57 colorectal tumour samples and an equal number of adjacent normal tissues. A high frequency of BRAFV600E mutation 21% (12/57) was identified in tumour tissues by MASMP compared to only 5.2% by direct DNA sequencing. Statistical analysis indicated that compared to BRAF-negative colorectal tumours, patients with BRAFV600E colorectal tumours were more likely to be >50 years old (61%) (P < 0.03). These tumours were more likely to be of clinical tumour stages III and IV (63%) (P < 0.04) with lymph node metastasis (52%) (P < 0.02) and characterised by a high-grade histology (63%) (P < 0.04). Colorectal patients harbouring BRAFV600E mutation experience more relapse/recurrence (52%) (P < 0.02). We, therefore, conclude that BRAFV600E mutation can be used as an indicator of poor prognosis to predict the outcome for CRC patients from Kashmir. MASMP proved to be a simple, sensitive and reliable technique for screening patients for BRAFV600E mutation. Testing for this mutation may be useful for selecting initial therapy mode and for follow-up monitoring in CRC patients.

Long-term outcomes of early compared to late onset choledochocholedochal anastomotic strictures after orthotopic liver transplantation.

BACKGROUND: Endoscopic treatment of anastomotic biliary stricture (ABS) after liver transplantation (LT) has been proven to be effective and safe, but long-term outcomes of early compared to late onset ABS have not been studied. The aim of this study is to compare the long-term outcome of early ABS to late ABS. METHODS: Of the 806 adult LT recipients (04/2006-12/2012), 93 patients met the criteria for inclusion, and were grouped into non-ABS (no stenosis on ERCP, n=41), early ABS (stenosis <90 days after LT, 18 [19.3%]), and late ABS (stenosis ≥90 days after LT, 34 [36.5%]). A propensity matched control group for the ABS group (n=42) was obtained matched for outcome variables for age, gender, and calculated MELD score at listing. RESULTS: Mean number of ERCPs (2.33±1.3 vs 2.56±1.5, P=.69) were comparable between the groups; however, significantly better long-term resolution of the stricture was noted in the early ABS group (94.44% vs 67.65%, P=.04). Kaplan-Meier analysis revealed worst survival in the early ABS group compared to the non-ABS, late ABS, and control groups (P=.0001). CONCLUSION: LT recipients with early ABS have inferior graft survival despite better response to endoscopic intervention.

Targeting Neurotrophins to Specific Populations of Neurons: NGF, BDNF, and NT-3 and Their Relevance for Treatment of Spinal Cord Injury.

Neurotrophins are a family of proteins that regulate neuronal survival, synaptic function, and neurotransmitter release, and elicit the plasticity and growth of axons within the adult central and peripheral nervous system. Since the 1950s, these factors have been extensively studied in traumatic injury models. Here we review several members of the classical family of neurotrophins, the receptors they bind to, and their contribution to axonal regeneration and sprouting of sensory and motor pathways after spinal cord injury (SCI). We focus on nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), and their effects on populations of neurons within diverse spinal tracts. Understanding the cellular targets of neurotrophins and the responsiveness of specific neuronal populations will allow for the most efficient treatment strategies in the injured spinal cord.

Transfer of HMW glutenin subunits from Aegilops kotschyi to wheat through radiation hybridization.

High molecular weight glutenin subunits (HMWGS) are responsible for dough elasticity and bread making quality of bread wheat. Related wild non-progenitor species, Aegilops kotschyi possesses higher molecular weight x and y glutenin subunits than the bread wheat cultivars. A wheat-Aegilops substitution line with 1U chromosome was used for the transfer of (HMWGS) of 1U to wheat by using pollen radiation hybridization approach. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiling showed different patterns of allelic variations with either the presence or absence of HMWGS, Glu-1A (1, null), Glu-1B (7, 7 + 8, 17 + 18) and Glu-1D (5 + 10, 2 + 12, null). The pollen irradiated wheat-Aegilops derivatives, B-56-1-4-2, B-56-1-4-3, B-14-1 and B-14-2 with Glu1Ux and 1Uy and absence or presence of some Glu-1A and Glu-1B HMWGS showed high micro SDS sedimentation test (MST) values while B-16-1 and B-16-2 had moderate MST values and high protein content. However, B-58-3 with transfer of Glu-1Ux + 1Uy for Glu-1D showed very low MST values indicating that Glu-1Ux + 1Uy enhance MST value only in the presence of Glu1D HMWGS. The transfer/substitution of alien HMW-GS for Glu-1A and or Glu-1B loci only can lead to improved bread making quality of wheat.

Transfer of useful variability of high grain iron and zinc from Aegilops kotschyi into wheat through seed irradiation approach.

PURPOSE: To transfer the 2S chromosomal fragment(s) of Aegilops kotschyi (2S(k)) into the bread wheat genome which could lead to the biofortification of wheat with high grain iron and zinc content. MATERIALS AND METHODS: Wheat-Ae. kotschyi 2A/2S(k) substitution lines with high grain iron and zinc content were used to transfer the gene/loci for high grain Fe and Zn content into wheat using seed irradiation approach. RESULTS: Bread wheat plants derived from 40 krad-irradiated seeds showed the presence of univalents and multivalents during meiotic metaphase-I. Genomic in situ hybridization analysis of seed irradiation hybrid F2 seedlings showed several terminal and interstitial signals indicated the introgression of Ae. kotschyi chromosome segments. This proves the efficacy of seed radiation hybrid approach in gene transfer experiments. All the radiation-treated hybrid plants with high grain Fe and Zn content were analyzed with wheat group 2 chromosome-specific polymorphic simple sequence repeat markers to identify the introgression of small alien chromosome fragment(s). CONCLUSION: Radiation-induced hybrids showed more than 65% increase in grain iron and 54% increase in Zn contents with better harvest index than the elite wheat cultivar WL711 indicating effective and compensating translocations of 2S(k) fragments into wheat genome.

Optical coherence tomography (OCT) evaluation of intermediate coronary lesions in patients with NSTEMI.

INTRODUCTION: Coronary angiography is commonly performed following non-ST segment elevation myocardial infarction (NSTEMI) to assess the need for revascularization. Some of these patients have myocardial infarction (MI) with no obstructive coronary atherosclerosis (MINOCA). Patients without severe obstructive lesions are usually treated conservatively. However, coronary angiography has known limitations in the assessment of lesion severity. We report our experience of using coronary Optical Coherence Tomography (OCT) in a series of patients without severe obstructive coronary lesions. METHODS: 165 patients underwent coronary OCT at Vancouver General Hospital. NSTEMI was the clinical presentation in 70 patients and 26 had angiographically intermediate lesions with 40%-69% diameter stenosis. Prior to OCT image acquisition, intracoronary nitroglycerin 100-200µg was administered. Blood in the vessel was displaced using contrast media by manual injections. RESULTS: OCT of the angiographically intermediate lesions showed larger minimal luminal area (MLA) than the angiographically severe lesions (MLA 3.3mm(2)±1.8mm(2) vs. 1.6mm(2)±0.6mm(2), p<0.001) and less severe % lumen area stenosis (54.2%±11.4% vs. 70.9%±6.8%, p=0.001). Plaque rupture or intracoronary thrombus was detected in 8/26 (31%) patients. PCI with stent deployment was performed in 16 patients (62%). CONCLUSION: In stabilized patients with NSTEMI and angiographically intermediate disease, OCT examination confirmed the lack of severe anatomical stenosis in most patients. However, OCT also identified coronary lesions with unstable features. Further research is needed to help guide management of this subgroup of patients.

Consumer use of over-the-counter proton pump inhibitors in patients with gastroesophageal reflux disease.

OBJECTIVES: Optimal administration of proton pump inhibitor (PPI) for the treatment of gastroesophageal reflux disease (GERD) requires consideration of meal timing. Since becoming available over the counter (OTC), no studies have assessed treatment patterns and symptom control in OTC consumers. The objective of this study was to survey dosing patterns and symptom control in OTC and prescription PPI users. METHODS: Patients at five clinics were surveyed regarding diagnosis of GERD, use of OTC or prescription PPIs, information on time of day dosing, demographics, and Gastroesophageal Reflux Disease Symptom Assessment Scale (GSAS; 2001, Johnson & Johnson). RESULTS: Of the 1,959 patients surveyed, 610 (31%) used PPIs for GERD. Of these, 190 (31%) and 223 (37%) received prescriptions from gastroenterologists (GIs) and primary care physicians (PCPs), respectively; 197 (32%) purchased OTC PPIs. Of the patients prescribed PPIs by GIs, 71% were optimal users, whereas 47% of patients receiving prescriptions from PCPs and 39% of consumers used PPIs optimally (P<0.001 compared with GIs). GSAS symptom, frequency, and severity scores were significantly better in patients prescribed PPIs by GIs (all P<0.001, GI compared with PCP and consumer). GSAS symptom, frequency, and severity scores were also significantly better in patients using PPIs optimally (P<0.001 for all parameters) compared with those taking PPIs suboptimally or excessively. CONCLUSIONS: Patients receiving prescription PPI from a GI are more likely to be optimal users with better symptom control. Conversely, consumers are more likely to be suboptimal users with inadequate symptom control.

The role of nonsteroidal anti-inflammatory drugs in the prevention of post endoscopic retrograde cholangiopancreatography pancreatitis.

Post-ERCP pancreatitis (PEP) is the most common major complication associated with ERCP. Beginning with an overview of the risk factors for the development of PEP, this review introduces the mechanism of injury in PEP and the role of pharmacological prevention. NSAIDs are increasingly found to offer prevention against the development of PEP, and their mechanism and supportive data are summarized, especially in relationship to the practice of prophylactic pancreatic duct stenting.

Ketamine reverses stress-induced depression-like behavior and increased GABA levels in the anterior cingulate: an 11.7 T 1H-MRS study in rats.

Gamma-aminobutyric acid (GABA) is the major inhibitory amino acid neurotransmitter in the brain and is primarily responsible for modulating excitatory tone. Clinical neuroimaging studies show decreased GABA levels in the anterior cingulate of patients with mood disorders, including major depressive disorder. Chronic unpredictable stress (CUS) is an animal model thought to mimic the stressful events that may precipitate clinical depression in humans. In this study male Sprague-Dawley rats were subjected to a modified CUS paradigm that used a random pattern of unpredictable stressors twice daily for 10 days to explore the early developmental stages of depression-like endophenotypes. Control rats were handled daily for 10 days. Some rats from each treatment group received an injection of ketamine (40 mg/kg) after the final stressor. One day following the final stressor rats were tested for behavioral effects in the forced swim test and then euthanized to collect trunk blood and anterior cingulate brain samples. GABA levels were measured in anterior cingulate samples ex vivo using proton magnetic resonance spectroscopy ((1)H-MRS) at 11.7 T. Animals subjected to CUS had lower body weights, higher levels of blood corticosterone, and increased immobility in the forced swim test; all of which suggest that the stress paradigm induced a depression-like phenotype. GABA levels in the anterior cingulate were significantly increased in the stressed animals compared to controls. Administration of ketamine on the last day of treatment blunted the depression-like behavior and increased GABA levels in the anterior cingulate following CUS. These data indicate that stress disrupts GABAergic signaling, which may over time lead to symptoms of depression and ultimately lower basal levels of cortical (1)H-MRS GABA that are seen in humans with depression. Furthermore, the data suggests that ketamine modulates cortical GABA levels as a mechanism of its antidepressant activity.

Novel C-1 substituted cocaine analogs unlike cocaine or benztropine.

Despite a wealth of information on cocaine-like compounds, there is no information on cocaine analogs with substitutions at C-1. Here, we report on (R)-(-)-cocaine analogs with various C-1 substituents: methyl (2), ethyl (3), n-propyl (4), n-pentyl (5), and phenyl (6). Analog 2 was equipotent to cocaine as an inhibitor of the dopamine transporter (DAT), whereas 3 and 6 were 3- and 10-fold more potent, respectively. None of the analogs, however, stimulated mouse locomotor activity, in contrast to cocaine. Pharmacokinetic assays showed compound 2 occupied mouse brain rapidly, as cocaine itself; moreover, 2 and 6 were behaviorally active in mice in the forced-swim test model of depression and the conditioned place preference test. Analog 2 was a weaker inhibitor of voltage-dependent Na+ channels than cocaine, although 6 was more potent than cocaine, highlighting the need to assay future C-1 analogs for this activity. Receptorome screening indicated few significant binding targets other than the monoamine transporters. Benztropine-like "atypical" DAT inhibitors are known to display reduced cocaine-like locomotor stimulation, presumably by their propensity to interact with an inward-facing transporter conformation. However, 2 and 6, like cocaine, but unlike benztropine, exhibited preferential interaction with an outward-facing conformation upon docking in our DAT homology model. In summary, C-1 cocaine analogs are not cocaine-like in that they are not stimulatory in vivo. However, they are not benztropine-like in binding mechanism and seem to interact with the DAT similarly to cocaine. The present data warrant further consideration of these novel cocaine analogs for antidepressant or cocaine substitution potential.