Browsing by Author "Khanna, Savita"

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    Ad hoc Hybrid Synaptic Junctions to Detect Nerve Stimulation and its Application to Detect Onset of Diabetic Polyneuropathy
    (Elsevier, 2020-12) Gupta, Sujasha; Ghatak, Subhadip; Hery, Travis; Khanna, Savita; El-Masry, Mohamed; Sundaresan, Vishnu Baba; Sen, Chandan K.; Surgery, School of Medicine
    We report a minimally invasive, synaptic transistor-based construct to monitor in vivo neuronal activity via a longitudinal study in mice and use depolarization time from measured data to predict the onset of polyneuropathy. The synaptic transistor is a three-terminal device in which ionic coupling between pre- and post-synaptic electrodes provides a framework for sensing low-power (sub μW) and high-bandwidth (0.1–0.5kHz) ionic currents. A validated first principles-based approach is discussed to demonstrate the significance of this sensing framework and we introduce a metric, referred to as synaptic efficiency to quantify structural and functional properties of the electrodes in sensing. The application of this framework for in vivo neuronal sensing requires a post-synaptic electrode and its reference electrode and the tissue becomes the pre-synaptic signal. The ionic coupling resembles axo-axonic junction and hence we refer to this framework as an ad hoc synaptic junction. We demonstrate that this arrangement can be applied to measure excitability of sciatic nerves due to a stimulation of the footpad in cohorts of m+/db and db/db mice for detecting loss in sensitivity and onset of polyneuropathy. The signal attributes were subsequently integrated with machine learning-based framework to identify the probability of polyneuropathy and to detect the onset of diabetic polyneuropathy.
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    Collagenase-based wound debridement agent induces extracellular matrix supporting phenotype in macrophages
    (Springer Nature, 2024-02-08) Banerjee, Pradipta; Das, Amitava; Singh, Kanhaiya; Khanna, Savita; Sen, Chandan K.; Roy, Sashwati; Surgery, School of Medicine
    Macrophages assume diverse phenotypes and functions in response to cues from the microenvironment. Earlier we reported an anti-inflammatory effect of Collagenase Santyl® Ointment (CSO) and the active constituent of CSO (CS-API) on wound macrophages in resolving wound inflammation indicating roles beyond debridement in wound healing. Building upon our prior finding, this study aimed to understand the phenotypes and subsets of macrophages following treatment with CS-API. scRNA-sequencing was performed on human blood monocyte-derived macrophages (MDM) following treatment with CS-API for 24 h. Unbiased data analysis resulted in the identification of discrete macrophage subsets based on their gene expression profiles. Following CS-API treatment, clusters 3 and 4 displayed enrichment of macrophages with high expression of genes supporting extracellular matrix (ECM) function. IPA analysis identified the TGFβ-1 pathway as a key hub for the CS-API-mediated ECM-supportive phenotype of macrophages. Earlier we reported the physiological conversion of wound-site macrophages to fibroblasts in granulation tissue and impairment of such response in diabetic wounds, leading to compromised ECM and tensile strength. The findings that CSO can augment the physiological conversion of macrophages to fibroblast-like cells carry significant clinical implications. This existing clinical intervention, already employed for wound care, can be readily repurposed to improve the ECM response in chronic wounds.
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    Cutaneous Epithelial to Mesenchymal Transition Activator ZEB1 Regulates Wound Angiogenesis and Closure in a Glycemic Status–Dependent Manner
    (American Diabetes Association, 2019-11) Singh, Kanhaiya; Sinha, Mithun; Pal, Durba; Tabasum, Saba; Gnyawali, Surya C.; Khona, Dolly; Sarkar, Subendu; Mohanty, Sujit K.; Soto-Gonzalez, Fidel; Khanna, Savita; Roy, Sashwati; Sen, Chandan K.; Surgery, School of Medicine
    Epithelial to mesenchymal transition (EMT) and wound vascularization are two critical interrelated processes that enable cutaneous wound healing. Zinc finger E-box binding homeobox 1 (ZEB1), primarily studied in the context of tumor biology, is a potent EMT activator. ZEB1 is also known to contribute to endothelial cell survival as well as stimulate tumor angiogenesis. The role of ZEB1 in cutaneous wounds was assessed using Zeb1+/− mice, as Zeb1−/− mice are not viable. Quantitative stable isotope labeling by amino acids in cell culture (SILAC) proteomics was used to elucidate the effect of elevated ZEB1, as noted during hyperglycemia. Under different glycemic conditions, ZEB1 binding to E-cadherin promoter was investigated using chromatin immunoprecipitation. Cutaneous wounding resulted in loss of epithelial marker E-cadherin with concomitant gain of ZEB1. The dominant proteins downregulated after ZEB1 overexpression functionally represented adherens junction pathway. Zeb1+/− mice exhibited compromised wound closure complicated by defective EMT and poor wound angiogenesis. Under hyperglycemic conditions, ZEB1 lost its ability to bind E-cadherin promoter. Keratinocyte E-cadherin, thus upregulated, resisted EMT required for wound healing. Diabetic wound healing was improved in ZEB+/− as well as in db/db mice subjected to ZEB1 knockdown. This work recognizes ZEB1 as a key regulator of cutaneous wound healing that is of particular relevance to diabetic wound complication.
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    Driving adult tissue repair via re-engagement of a pathway required for fetal healing
    (Elsevier, 2023) Ghatak, Subhadip; Khanna, Savita; Roy, Sashwati; Thirunavukkarasu, Mahesh; Pradeep, Seetur R.; Wulff, Brian C.; El Masry, Mohamed S.; Sharma, Anu; Palakurti, Ravichand; Ghosh, Nandini; Xuan, Yi; Wilgus, Traci A.; Maulik, Nilanjana; Yoder, Mervin C.; Sen, Chandan K.; Surgery, School of Medicine
    Fetal cutaneous wound closure and repair differ from that in adulthood. In this work, we identify an oxidant stress sensor protein, nonselenocysteine-containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), that is abundantly expressed in normal fetal epidermis (and required for fetal wound closure), though not in adult epidermis, but is variably re-induced upon adult tissue wounding. NPGPx is a direct target of the miR-29 family. Following injury, abundance of miR-29 is lowered, permitting a prompt increase in NPGPx transcripts and protein expression in adult wound-edge tissue. NPGPx expression was required to mediate increased keratinocyte migration induced by miR-29 inhibition in vitro and in vivo. Increased NPGPx expression induced increased SOX2 expression and β-catenin nuclear localization in keratinocytes. Augmenting physiologic NPGPx expression via experimentally induced miR-29 suppression, using cutaneous tissue nanotransfection or targeted lipid nanoparticle delivery of anti-sense oligonucleotides, proved to be sufficient to overcome the deleterious effects of diabetes on this specific pathway to enhance tissue repair.
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    Electroceutical fabric lowers zeta potential and eradicates coronavirus infectivity upon contact
    (Nature, 2021-12) Ghatak, Subhadip; Khona, Dolly K.; Sen, Abhishek; Huang, Kaixiang; Jagdale, Gargi; Singh, Kanhaiya; Gopalakrishnan, Vinoj; Cornetta, Kenneth G.; Roy, Sashwati; Khanna, Savita; Baker, Lane A.; Sen, Chandan K.; Medical and Molecular Genetics, School of Medicine
    Coronavirus with intact infectivity attached to PPE surfaces pose significant threat to the spread of COVID-19. We tested the hypothesis that an electroceutical fabric, generating weak potential difference of 0.5 V, disrupts the infectivity of coronavirus upon contact by destabilizing the electrokinetic properties of the virion. Porcine respiratory coronavirus AR310 particles (105) were placed in direct contact with the fabric for 1 or 5 min. Following one minute of contact, zeta potential of the porcine coronavirus was significantly lowered indicating destabilization of its electrokinetic properties. Size-distribution plot showed appearance of aggregation of the virus. Testing of the cytopathic effects of the virus showed eradication of infectivity as quantitatively assessed by PI-calcein and MTT cell viability tests. This work provides the rationale to consider the studied electroceutical fabric, or other materials with comparable property, as material of choice for the development of PPE in the fight against COVID-19.
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    Electroceutical Fabric Lowers Zeta Potential and Eradicates Coronavirus Infectivity upon Contact
    (2020-05-15) Sen, Abhishek; Khona, Dolly; Ghatak, Subhadip; Gopalakrishnan, Vinoj; Cornetta, Kenneth; Roy, Sashwati; Khanna, Savita; Sen, Chandan; Surgery, School of Medicine
    Coronavirus with intact infectivity attached to PPE surfaces pose significant threat to the spread of COVID-19. We tested the hypothesis that an electroceutical fabric, generating weak potential difference of 0.5V, disrupts the infectivity of coronavirus upon contact by destabilizing the electrokinetic properties of the virion. Respiratory coronavirus particles (105) were placed in direct contact with the fabric for 1 or 5 minutes. Viral particles (2.5-4x104) were recovered from the fabric. Following one minute of contact, zeta potential of the coronavirus was significantly lowered indicating destabilization of its electrokinetic properties. Size-distribution plot showed appearance of aggregation of the virus. Testing of the cytopathic effects of the virus showed eradication of infectivity as quantitatively assessed by PI-calcein and MTT cell viability tests. This work provides the rationale to consider the studied electroceutical fabric, or other materials with comparable property, as material of choice for the development of PPE in the fight against COVID-19.
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    Endothelial Phospholipase Cγ2 Improves Outcomes of Diabetic Ischemic Limb Rescue Following VEGF Therapy
    (American Diabetes Association, 2022) Rustagi, Yashika; Abouhashem, Ahmed S.; Verma, Priyanka; Verma, Sumit S.; Hernandez, Edward; Liu, Sheng; Kumar, Manishekhar; Guda, Poornachander R.; Srivastava, Rajneesh; Mohanty, Sujit K.; Kacar, Sedat; Mahajan, Sanskruti; Wanczyk, Kristen E.; Khanna, Savita; Murphy, Michael P.; Gordillo, Gayle M.; Roy, Sashwati; Wan, Jun; Sen, Chandan K.; Singh, Kanhaiya; Medicine, School of Medicine
    Therapeutic vascular endothelial growth factor (VEGF) replenishment has met with limited success for the management of critical limb-threatening ischemia. To improve outcomes of VEGF therapy, we applied single-cell RNA sequencing (scRNA-seq) technology to study the endothelial cells of the human diabetic skin. Single-cell suspensions were generated from the human skin followed by cDNA preparation using the Chromium Next GEM Single-cell 3' Kit v3.1. Using appropriate quality control measures, 36,487 cells were chosen for downstream analysis. scRNA-seq studies identified that although VEGF signaling was not significantly altered in diabetic versus nondiabetic skin, phospholipase Cγ2 (PLCγ2) was downregulated. The significance of PLCγ2 in VEGF-mediated increase in endothelial cell metabolism and function was assessed in cultured human microvascular endothelial cells. In these cells, VEGF enhanced mitochondrial function, as indicated by elevation in oxygen consumption rate and extracellular acidification rate. The VEGF-dependent increase in cell metabolism was blunted in response to PLCγ2 inhibition. Follow-up rescue studies therefore focused on understanding the significance of VEGF therapy in presence or absence of endothelial PLCγ2 in type 1 (streptozotocin-injected) and type 2 (db/db) diabetic ischemic tissue. Nonviral topical tissue nanotransfection technology (TNT) delivery of CDH5 promoter-driven PLCγ2 open reading frame promoted the rescue of hindlimb ischemia in diabetic mice. Improvement of blood flow was also associated with higher abundance of VWF+/CD31+ and VWF+/SMA+ immunohistochemical staining. TNT-based gene delivery was not associated with tissue edema, a commonly noted complication associated with proangiogenic gene therapies. Taken together, our study demonstrates that TNT-mediated delivery of endothelial PLCγ2, as part of combination gene therapy, is effective in diabetic ischemic limb rescue.
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    Fabrication and use of silicon hollow-needle arrays to achieve tissue nanotransfection in mouse tissue in vivo
    (Springer Nature, 2021) Xuan, Yi; Ghatak, Subhadip; Clark, Andrew; Li, Zhigang; Khanna, Savita; Pak, Dongmin; Agarwal, Mangilal; Roy, Sashwati; Duda, Peter; Sen, Chandan K.; Surgery, School of Medicine
    Tissue nanotransfection (TNT) is an electromotive gene transfer technology that was developed to achieve tissue reprogramming in vivo. This protocol describes how to fabricate the required hardware, commonly referred to as a TNT chip, and use it for in vivo TNT. Silicon hollow-needle arrays for TNT applications are fabricated in a standardized and reproducible way. In <1 s, these silicon hollow-needle arrays can be used to deliver plasmids to a predetermined specific depth in murine skin in response to pulsed nanoporation. Tissue nanotransfection eliminates the need to use viral vectors, minimizing the risk of genomic integration or cell transformation. The TNT chip fabrication process typically takes 5-6 d, and in vivo TNT takes 30 min. This protocol does not require specific expertise beyond a clean room equipped for basic nanofabrication processes.
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    Genome-wide DNA hypermethylation opposes healing in patients with chronic wounds by impairing epithelial-mesenchymal transition
    (The American Society for Clinical Investigation, 2022) Singh, Kanhaiya; Rustagi, Yashika; Abouhashem, Ahmed S.; Tabasum, Saba; Verma, Priyanka; Hernandez, Edward; Pal, Durba; Khona, Dolly K.; Mohanty, Sujit K.; Kumar, Manishekhar; Srivastava, Rajneesh; Guda, Poornachander R.; Verma, Sumit S.; Mahajan, Sanskruti; Killian, Jackson A.; Walker, Logan A.; Ghatak, Subhadip; Mathew-Steiner, Shomita S.; Wanczyk, Kristen E.; Liu, Sheng; Wan, Jun; Yan, Pearlly; Bundschuh, Ralf; Khanna, Savita; Gordillo, Gayle M.; Murphy, Michael P.; Roy, Sashwati; Sen, Chandan K.; Surgery, School of Medicine
    An extreme chronic wound tissue microenvironment causes epigenetic gene silencing. An unbiased whole-genome methylome was studied in the wound-edge tissue of patients with chronic wounds. A total of 4,689 differentially methylated regions (DMRs) were identified in chronic wound-edge skin compared with unwounded human skin. Hypermethylation was more frequently observed (3,661 DMRs) in the chronic wound-edge tissue compared with hypomethylation (1,028 DMRs). Twenty-six hypermethylated DMRs were involved in epithelial-mesenchymal transition (EMT). Bisulfite sequencing validated hypermethylation of a predicted specific upstream regulator TP53. RNA-Seq analysis was performed to qualify findings from methylome analysis. Analysis of the downregulated genes identified the TP53 signaling pathway as being significantly silenced. Direct comparison of hypermethylation and downregulated genes identified 4 genes, ADAM17, NOTCH, TWIST1, and SMURF1, that functionally represent the EMT pathway. Single-cell RNA-Seq studies revealed that these effects on gene expression were limited to the keratinocyte cell compartment. Experimental murine studies established that tissue ischemia potently induces wound-edge gene methylation and that 5'-azacytidine, inhibitor of methylation, improved wound closure. To specifically address the significance of TP53 methylation, keratinocyte-specific editing of TP53 methylation at the wound edge was achieved by a tissue nanotransfection-based CRISPR/dCas9 approach. This work identified that reversal of methylation-dependent keratinocyte gene silencing represents a productive therapeutic strategy to improve wound closure.
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    Hydrolyzed Collagen Powder Dressing Improves Wound Inflammation, Perfusion, and Breaking Strength of Repaired Tissue
    (Liebert, 2023) Kumar, Manishekhar; Banerjee, Pradipta; Das, Amitava; Singh, Kanhaiya; Guith, Tanner; Kacar, Sedat; Gourishetti, Karthik; Sen, Chandan K.; Roy, Sashwati; Khanna, Savita; Surgery, School of Medicine
    Hydrolyzed collagen-based matrices are widely used as wound care dressings. Information on the mechanism of action of such dressings is scanty. The objective of this study was to test the effect of a specific hydrolyzed collagen powder (HCP), which is extensively used for wound care management in the United States. The effects of HCP on resolution of wound inflammation, perfusion, closure, and breaking strength of the repaired skin were studied in an experimental murine model. In early (day 7) inflammatory phase of wound macrophages, HCP treatment boosted phagocytosis and efferocytosis of wound-site macrophages. In these cells, inducible reactive oxygen species were also higher on day (d) 7. HCP treatment potentiated the expression of anti-inflammatory interleukin (IL)-10 cytokine and proangiogenic vascular endothelial growth factor (VEGF) production. Excisional wounds dressed with HCP showed complete closure on day 21, while the control wounds remained open. HCP treatment also demonstrated improved quality of wound healing as marked by the improved breaking strength of the closed wound tissue/repaired skin. These data represent first evidence on the mechanism of action of clinically used HCP. HCP dressing favorably influenced both wound inflammation and vascularization. Improved breaking strength of HCP-treated repaired skin lays the rationale for future studies testing the hypothesis that HCP-treated closed wounds would show fewer recurrences.