Genetic reprogramming with stem cells regenerates glomerular epithelial podocytes in Alport syndrome.

Glomerular filtration relies on the type IV collagen (ColIV) network of the glomerular basement membrane, namely, in the triple helical molecules containing the α3, α4, and α5 chains of ColIV. Loss of function mutations in the genes encoding these chains (Col4a3, Col4a4, and Col4a5) is associated with the loss of renal function observed in Alport syndrome (AS). Precise understanding of the cellular basis for the patho-mechanism remains unknown and a specific therapy for this disease does not currently exist. Here, we generated a novel allele for the conditional deletion of Col4a3 in different glomerular cell types in mice. We found that podocytes specifically generate α3 chains in the developing glomerular basement membrane, and that its absence is sufficient to impair glomerular filtration as seen in AS. Next, we show that horizontal gene transfer, enhanced by TGFß1 and using allogenic bone marrow-derived mesenchymal stem cells and induced pluripotent stem cells, rescues Col4a3 expression and revive kidney function in Col4a3-deficient AS mice. Our proof-of-concept study supports that horizontal gene transfer such as cell fusion enables cell-based therapy in Alport syndrome.

Management of frontal sinus trauma: a retrospective study of surgical interventions and complications.

BACKGROUND: Frontal sinus injuries are relatively rare among facial bone traumas. Without proper treatment, they can lead to fatal intracranial complications, including meningitis or brain abscesses, as well as aesthetic and functional sequelae. The management of frontal sinus injuries remains controversial, with various treatment methods and outcomes being reported. This article describes the clinical characteristics, surgical methods, and outcomes among 17 patients who underwent surgery for frontal sinus injury and related complications. CASE PRESENTATION: We retrospectively included 17 patients who underwent surgery for frontal sinus injury and its related complications at the Kangwon National University Hospital between July 2010 and September 2021. Among them, six underwent simple open reduction and fixation of the anterior wall, eight underwent sinus obliteration, and three underwent cranialization. Two patients who underwent sinus obliteration died due to infection-related complications. The patient who underwent cranialization reported experiencing chronic headache and expressed dissatisfaction regarding the esthetic outcomes of the forehead. Except for these three patients, the other patients achieved satisfactory esthetic and functional recovery. CONCLUSION: Active surgical management of frontal sinus injuries is often required owing to the various complications caused by these injuries; however, several factors, including the fracture type, clinical presentation, related craniomaxillofacial injury, and medical history, should be considered while formulating the treatment plan. Surgical treatment through the opening of the frontal sinus should be actively considered in patients with severely damaged posterior wall fractures and those at risk of developing infection.

Tubulysins are Essential for the Preying of Ciliates by Myxobacteria.

Tubulysins are bioactive secondary metabolites produced by myxobacteria that promote microtubule disassembly. Microtubules are required for protozoa such as Tetrahymena to form cilia and flagella. To study the role of tubulysins in myxobacteria, we co-cultured myxobacteria and Tetrahymena. When 4000 Tetrahymena thermophila and 5.0 × 108 myxobacteria were added to 1 ml of CYSE medium and co-cultured for 48 h, the population of T. thermophila increased to more than 75,000. However, co-culturing tubulysin-producing myxobacteria, including Archangium gephyra KYC5002, with T. thermophila caused the population of T. thermophila to decrease from 4000 to less than 83 within 48 h. Almost no dead bodies of T. thermophila were observed in the culture medium. Co-culturing of T. thermophila and the A. gephyra KYC5002 strain with inactivation of the tubulysin biosynthesis gene led to the population of T. thermophila increasing to 46,667. These results show that in nature, most myxobacteria are preyed upon by T. thermophila, but some myxobacteria prey on and kill T. thermophila using tubulysins. Adding purified tubulysin A to T. thermophila changed the cell shape from ovoid to spherical and caused cell surface cilia to disappear.

Acquired αSMA Expression in Pericytes Coincides with Aberrant Vascular Structure and Function in Pancreatic Ductal Adenocarcinoma.

The subpopulations of tumor pericytes undergo pathological phenotype switching, affecting their normal function in upholding structural stability and cross-communication with other cells. In the case of pancreatic ductal adenocarcinoma (PDAC), a significant portion of blood vessels are covered by an α-smooth muscle actin (αSMA)-expressing pericyte, which is normally absent from capillary pericytes. The DesminlowαSMAhigh phenotype was significantly correlated with intratumoral hypoxia and vascular leakiness. Using an in vitro co-culture system, we demonstrated that cancer cell-derived exosomes could induce ectopic αSMA expression in pericytes. Exosome-treated αSMA+ pericytes presented altered pericyte markers and an acquired immune-modulatory feature. αSMA+ pericytes were also linked to morphological and biomechanical changes in the pericyte. The PDAC exosome was sufficient to induce αSMA expression by normal pericytes of the healthy pancreas in vivo, and the vessels with αSMA+ pericytes were leaky. This study demonstrated that tumor pericyte heterogeneity could be dictated by cancer cells, and a subpopulation of these pericytes confers a pathological feature.

Single-Molecule Force Probing of RGD-Binding Integrins on Pancreatic Cancer Cells.

Integrin-targeting arginine-glycine-aspartic acid (RGD)-based nanocarriers have been widely used for tumor imaging, monitoring of tumor development, and delivery of anticancer drugs. However, the thermodynamics of an RGD-integrin formation and dissociation associated with binding dynamics, affinity, and stability remains unclear. Here, we probed the binding strength of the binary complex to live pancreatic cancer cells using single-molecule binding force spectroscopy methods, in which RGD peptides were functionalized on a force probe tip through poly(ethylene glycol) (PEG)-based bifunctional linker molecules. While the density of integrin αV receptors on the cell surface varies more than twofold from cell line to cell line, the individual RGD-integrin complexes exhibited a cell type-independent, monovalent bond strength. The load-dependent bond strength of multivalent RGD-integrin interactions scaled sublinearly with increasing bond number, consistent with the noncooperative, parallel bond model. Furthermore, the multivalent bonds ruptured sequentially either by one or in multiples, and the force strength was comparable to the synchronous rupture force. Comparison of energy landscapes of the bond number revealed a substantial decrease of kinetic off-rates for multivalent bonds, along with the increased width of the potential well and the increased potential barrier height between bound and unbound states, enhancing the stability of the multivalent bonds between them.

Engineered exosomes targeting MYC reverse the proneural-mesenchymal transition and extend survival of glioblastoma.

Dysregulated Myc signaling is a key oncogenic pathway in glioblastoma multiforme (GBM). Yet, effective therapeutic targeting of Myc continues to be challenging. Here, we demonstrate that exosomes generated from human bone marrow mesenchymal stem cells (MSCs) engineered to encapsulate siRNAs targeting Myc (iExo-Myc) localize to orthotopic GBM tumors in mice. Treatment of late stage GBM tumors with iExo-Myc inhibits proliferation and angiogenesis, suppresses tumor growth, and extends survival. Transcriptional profiling of tumors reveals that the mesenchymal transition and estrogen receptor signaling pathways are impacted by Myc inhibition. Single nuclei RNA sequencing (snRNA-seq) shows that iExo-Myc treatment induces transcriptional repression of multiple growth factor and interleukin signaling pathways, triggering a mesenchymal to proneural transition and shifting the cellular landscape of the tumor. These data confirm that Myc is an effective anti-glioma target and that iExo-Myc offers a feasible, readily translational strategy to inhibit challenging oncogene targets for the treatment of brain tumors.

Spinal epidural abscess due to coinfection of bacteria and tuberculosis: A case report.

BACKGROUND: Spinal epidural abscess (SEA) is a rare condition that mostly results from infection with either bacteria or tuberculosis. However, coinfection with bacteria and tuberculosis is extremely rare, and it results in delays in diagnosis and antimicrobial treatment causing unfavorable outcomes. CASE SUMMARY: A 75-year-old female visited the hospital with low back pain, and magnetic resonance imaging (MRI) revealed an SEA at the lumbosacral segment. Staphylococcus hominis and methicillin-resistant Staphylococcus epidermidis were identified from preoperative blood culture and intraoperative abscess culture, respectively. Thus, the patient underwent treatment with vancomycin medication for 9 wk after surgical drainage of the SEA. However, the low back pain recurred 2 wk after vancomycin treatment. MRI revealed an aggravated SEA in the same area in addition to erosive destruction of vertebral bodies. Second surgery was performed for SEA removal and spinal instrumentation. The microbiological study and pathological examination confirmed Mycobacterium tuberculosis as the pathogen concurrent with the bacterial SEA. The patient improved completely after 12 mo of antitubercular medication. CONCLUSION: We believe that the identification of a certain pathogen in SEAs does not exclude coinfection with other pathogens. Tubercular coinfection should be suspected if an SEA does not improve despite appropriate antibiotics for the identified pathogen.

Dynamic cellular biomechanics in responses to chemotherapeutic drug in hypoxia probed by atomic force spectroscopy.

The changes in cellular structure play an important role in cancer cell development, progression, and metastasis. By exploiting single-cell, force spectroscopy methods, we probed biophysical and biomechanical kinetics (stiffness, morphology, roughness, adhesion) of brain, breast, prostate, and pancreatic cancer cells with standard chemotherapeutic drugs in normoxia and hypoxia over 12-24 hours. After exposure to the drugs, we found that brain, breast, and pancreatic cancer cells became approximately 55-75% less stiff, while prostate cancer cells became more stiff, due to either drug-induced disruption or reinforcement of cytoskeletal structure. However, the rate of the stiffness change decreased up to 2-folds in hypoxia, suggesting a correlation between cellular stiffness and drug resistance of cancer cells in hypoxic tumor microenvironment. Also, we observed significant changes in the cell body height, surface roughness, and cytoadhesion of cancer cells after exposure to drugs, which followed the trend of stiffness. Our results show that a degree of chemotherapeutic drug effects on biomechanical and biophysical properties of cancer cells is distinguishable in normoxia and hypoxia, which are correlated with alteration of cytoskeletal structure and integrity during drug-induced apoptotic process.

Methods and Techniques to Facilitate the Development of Clostridium novyi NT as an Effective, Therapeutic Oncolytic Bacteria.

The tumor microenvironment is characterized by anomalous vascularization, hypoxia, and acidity at the core of solid tumors that culminates in concentrated necrosis and immune system dysregulation among other effects. While this environment presents several challenges for the development of oncotherapeutics that deliver their activity via the enhanced permeability and retention (EPR) effect of the leaky blood vessels around a tumor, oncolytic bacteria, or a class of bacteria with a noted capacity to lyse solid tumors, are attracted to the very environment found at the center of solid tumors that confounds other therapeutics. It is this capacity that allows for a potent, active penetration from the tumor margins into the core, and subsequent colonization to facilitate lysis and immune reactivation. Clostridium novyi in particular has recently shown great promise in preclinical and clinical trials when administered directly to the tumor. These studies indicate that C. novyi is uniquely poised to effectively accomplish the long sought after "holy grail" of oncotherapeutics: selective tumor localization via intravenous delivery. This study reports the development of efficient methods that facilitate experimental work and therapeutic translation of C. novyi including the ability to work with this obligate micro-anaerobe on the benchtop. Additionally, this study seeks to utilize this newfound experimental flexibility to address several gaps in the current knowledge regarding the efficacy of CRIPSR/Cas9-mediated gene insertion in this species to further develop this oncolytic bacteria and the genetic customization of bacteria in general.

Type I collagen deletion in αSMA+ myofibroblasts augments immune suppression and accelerates progression of pancreatic cancer.

Stromal desmoplastic reaction in pancreatic ductal adenocarcinoma (PDAC) involves significant accumulation of type I collagen (Col1). However, the precise molecular and mechanistic contribution of Col1 in PDAC progression remains unknown. Activated pancreatic stellate cells/αSMA+ myofibroblasts are major contributors of Col1 in the PDAC stroma. We use a dual-recombinase genetic mouse model of spontaneous PDAC to delete Col1 specifically in myofibroblasts. This results in significant reduction of total stromal Col1 content and accelerates the emergence of PanINs and PDAC, decreasing overall survival. Col1 deletion leads to Cxcl5 upregulation in cancer cells via SOX9. Increase in Cxcl5 is associated with recruitment of myeloid-derived suppressor cells and suppression of CD8+ T cells, which can be attenuated with combined targeting of CXCR2 and CCR2 to restrain accelerated PDAC progression in the setting of stromal Col1 deletion. Our results unravel the fundamental role of myofibroblast-derived Co1l in regulating tumor immunity and restraining PDAC progression.

Clinical Characteristics of Spinal Epidural Abscess Accompanied by Bacteremia.

OBJECTIVE: The treatment of choice for spinal epidural abscess (SEA) generally is urgent surgery in combination with intravenous antibiotic treatment. However, the optimal duration of antibiotic treatment has not been established to date, although 4-8 weeks is generally advised. Moreover, some researchers have reported that bacteremia is a risk factor for failure of antibiotic treatment in SEA. In this study, we investigated the clinical characteristics of SEA accompanied by bacteremia and also determined whether the conventional 4-8 weeks of antibiotic treatment is sufficient. METHODS: We retrospectively reviewed the medical records and radiological data of 23 patients with bacterial SEA who underwent open surgery from March 2010 to April 2020. All patients had bacteremia preoperatively and underwent weeks of perioperative antibiotic treatments based on their identified organisms until all symptoms of infection disappeared. All patients underwent microbiological studies of peripheral blood, specimens from SEA and concomitant infections. The mean follow-up duration was 35.2 months, excluding three patients who died. RESULTS: The male : female ratio was 15 : 8, and the mean age was 68.9 years. The SEA most commonly involved the lumbar spinal segment (73.9%), and the mean size was 2.9 vertebral body lengths. Mean time periods of 8.4 days and 16.6 days were required from admission to diagnosis and from admission to surgery, respectively. Concomitant infections more frequently resulted in delayed diagnosis (p=0.032), masking the symptoms of SEA. Methicillin-sensitive Staphylococcus aureus was the most commonly identified pathogen in both blood and surgical specimens. Seventeen patients (73.9%) showed no deficits at the final follow-up. The overall antibiotic treatment duration was a mean of 66.6 days, excluding three patients who died. This duration was longer than the conventionally advised 4-8 weeks (p=0.010), and psoas or paraspinal abscess required prolonged duration of antibiotic treatment (p=0.038). CONCLUSION: SEA accompanied by bacteremia required a longer duration (>8 weeks) of antibiotic treatment. In addition, the diagnosis was more frequently delayed in patients with concomitant infections. The duration of antibiotic treatment should be extended for SEA with bacteremia, and a high index of suspicion is mandatory for early diagnosis, especially in patients with concomitant infections.

Traumatic Subarachnoid Hemorrhage Resulting from Posterior Communicating Artery Rupture.

Traumatic subarachnoid hemorrhage (SAH), a common finding following head trauma, is usually a benign condition with a favorable outcome, seldom requiring surgical intervention. Unlike nontraumatic aneurysmal SAH, most cases of traumatic SAH occur in the sulci of the cerebral convexities, and only rarely arise at the base of the brain. Basal traumatic SAH can be life-threatening and is primarily associated with rupture of vertebrobasilar arteries. We herein present a rare case of basal traumatic SAH resulting from rupture of the posterior communicating artery (PCoA). A 77-year-old male was taken to the emergency department in a semicomatose state. Upon arrival at emergency room, the patient had a Glasgow coma scale (GCS) score of 6 (E1M3V2), and the neurologic examination demonstrated no focal neurologic deficit. Although the trauma history was evident from abrasions and bruising on the face and chest, brain computed tomography (CT) demonstrated basal SAH, which is typical for nontraumatic aneurysmal SAH. Subsequent digital subtraction angiography (DSA) disclosed a traumatic rupture at the mid-portion of right PCoA and ongoing extravasation of contrast media. Despite emergent trapping of the right PCoA by endovascular surgery, the patient's clinical condition only minimally improved. The patient remained bed-ridden with stuporous mentality and persistent hydrocephalus. To the best of our knowledge, this is the first reported case of basal traumatic SAH originating from rupture of the PCoA. This case demonstrates that a meticulous vascular workup is mandatory for every patient with basal SAH, even though a trauma history is clear.

Targeting the Tumor Core: Hypoxia-Responsive Nanoparticles for the Delivery of Chemotherapy to Pancreatic Tumors.

In pancreatic ductal adenocarcinoma (PDAC), early onset of hypoxia triggers remodeling of the extracellular matrix, epithelial-to-mesenchymal transition, increased cell survival, the formation of cancer stem cells, and drug resistance. Hypoxia in PDAC is also associated with the development of collagen-rich, fibrous extracellular stroma (desmoplasia), resulting in severely impaired drug penetration. To overcome these daunting challenges, we created polymer nanoparticles (polymersomes) that target and penetrate pancreatic tumors, reach the hypoxic niches, undergo rapid structural destabilization, and release the encapsulated drugs. In vitro studies indicated a high cellular uptake of the polymersomes and increased cytotoxicity of the drugs under hypoxia compared to unencapsulated drugs. The polymersomes decreased tumor growth by nearly 250% and significantly increased necrosis within the tumors by 60% in mice compared to untreated controls. We anticipate that these polymer nanoparticles possess a considerable translational potential for delivering drugs to solid hypoxic tumors.

A rare case of delayed hypersensitivity reaction to metal ions secondary to a remnant pedicle screw fragment after spinal arthrodesis.

Delayed hypersensitivity reaction to metal ions is rare and has been mostly documented among patients undergoing total joint arthroplasty with metal-on-metal bearing surfaces. Nonetheless, to our knowledge, only a few cases associated with spinal arthrodesis have been reported in the literature. The aim of this case report was to describe the clinical features of a rare case of delayed-type hypersensitivity reaction to nickel that occurred after an extraordinarily long period of time (approximately 15 years) after spinal arthrodesis. Here, we present a 38-year-old patient who was referred to us with a diagnosis of delayed-type hypersensitivity reaction to nickel that was established by a skin patch test. The chief sign and symptoms of the patient were allergic contact dermatitis along with intractable, generalized itching that started several months ago. The medical history revealed that the patient had undergone spinal arthrodesis using rods and pedicle screws approximately 15 years ago, followed by revision surgery to remove the implants. Radiological examination showed a remnant pedicle screw fragment at the third lumbar vertebra that had broken off during the removal of implants nine years ago. However, the signs and symptoms of metal hypersensitivity started shortly before the patient was referred to our hospital. We decided to remove the screw fragment as allergic contact dermatitis was refractory to medical treatment. On postoperative day one, the itching was completely resolved, although the skin patch test remained positive.

Pericytes in Breast Cancer.

Breast cancer is a heterogeneous disease driven not only by evolutionally diverse cancer cell themselves but also by highly dynamic microenvironment. At the center of the tumor microenvironment, tumor vasculature plays multiple roles from supporting tumor growth to providing a route for metastasis to the distant organ sites. Blood vessels in breast cancer present with perfusion defects associated with vessel dilation, tortuosity, and poor perivascular coverage (Li et al., Ultrasound Med 32:1145-1155, 2013; Eberhard et al., Cancer Res 60:1388-1393, 2000; Cooke et al., Cancer Cell 21:66-81, 2012). Such abnormal vascular system is partly due to the morphological and molecular alteration of pericytes that is accompanied by a significant heterogeneity within the populations (Kim et al., JCI Insight 1:e90733, 2016). While pericytes are implicated for their controversial roles in breast cancer metastasis (Cooke et al., Cancer Cell 21:66-81, 2012; Gerhardt and Semb, J Mol Med (Berl) 86:135-144, 2008; Keskin et al., Cell Rep 10:1066-1081, 2015; Meng et al., Future Oncol 11:169-179, 2015; Xian et al., J Clin Invest 116:642-651, 2006), the impact of their heterogeneity on breast cancer progression, metastasis, intratumoral immunity, and response to chemotherapy are largely unknown. Due to the complexity of angiogenic programs of breast cancer, the anti-angiogenic or anti-vascular treatment has been mostly unsuccessful (Tolaney et al., Proc Natl Acad Sci U S A 112:14325-14330, 2015; Mackey et al., Cancer Treat Rev 38:673-688, 2012; Sledge, J Clin Oncol 33:133-135, 2015) and requires much in-depth knowledge on different components of tumor microenvironment and how these stromal cells are interacting and communicating to each other. Therefore, understanding pericyte heterogeneity and their differential functional contribution will shed light on new potential approaches to treat breast cancer.

Enlarging acute tentorial subdural hematoma evacuated by surgery.

Acute intracranial subdural hematomas (SDHs) of tentorial type generally pose no serious clinical threats, and unlike other variants of SDH, rarely require surgical intervention. Herein, we present an exceedingly rare case of tentorial SDH, marked by gradual enlargement and eventually calling for surgical evacuation. A 55-year-old man presented to the emergency department after sustaining head trauma. Initially, he was alert, fully oriented, and neurologically stable. Although computed tomography (CT) of the brain revealed an acute SDH scantily distributed along right tentorium, brain swelling or midline shift was negligible. On the following day, he became confused, but pupil size and light reflex remained normal. A follow-up CT scan showed considerable enlargement of the acute SDH, with midline shift. In a matter of hours, he deteriorated to a stuporous state, as the SDH enlarged even more. We performed a craniotomy and completely evacuated the SDH on an emergency basis. As a result, the midline shift improved, and he again became alert, soon recovering without any new neurologic deficit. This illustrative case demonstrates that even a tentorial SDH may ultimately deteriorate, forcing surgical evacuation. We, therefore, feel that close observation is mandatory for such events, even if the initial volume is small.

Exosomes as Drug Carriers for Cancer Therapy.

Exosomes, biological extracellular vesicles, have recently begun to find use in targeted drug delivery in solid tumor research. Ranging from 30-120 nm in size, exosomes are secreted from cells and isolated from bodily fluids. Exosomes provide a unique material platform due to their characteristics, including physical properties such as stability, biocompatibility, permeability, low toxicity, and low immunogenicity-all critical to the success of any nanoparticle drug delivery system. In addition to traditional chemotherapeutics, natural products and RNA have been encapsulated for the treatment of breast, pancreatic, lung, prostate cancers, and glioblastoma. This review discusses current research on exosomes for drug delivery to solid tumors.

Jejunal perforation as an unusual presentation of total colonic aganglionosis in a neonate: A case report.

INTRODUCTION: Neonatal intestinal perforation usually occurs at distal small bowel secondary to distal bowel obstruction. The aim of this report is to describe an unusual case of total colonic aganglionosis with an initial presentation of proximal jejunal perforation. PRESENTATION OF CASE: A male newborn presented with jejunal perforation on the fifth day of life and was treated by laparoscopic primary repair. Abdominal distention persisted postoperatively, and radiological examination revealed an obstruction near the terminal ileum. Laparotomy showed a transition zone 30-cm proximal to the ileocecal valve, and diverting ileostomy and appendiceal biopsy was performed. Permanent section demonstrated the complete absence of ganglion cells in the appendix and total colonic aganglionosis was strongly suspected. DISCUSSION: Contrary to the classic teaching, proximal bowel perforation can occur in case of far distal obstruction, and careful distal evaluation would direct more appropriate surgical treatment option. CONCLUSIONS: Total colonic aganglionosis can present as a proximal bowel perforation. Careful distal evaluation can provide diagnostic clues in cases of proximal intestinal perforation. Appendiceal biopsy is a reliable tool for evaluating suspected total colonic aganglionosis, but multiple colonic and rectal biopsies should be obtained to confirm the diagnosis.

Endoscopic Treatment of Intussusception due to Intestinal Tuberculosis.

Traditionally, adult intussusception has required a bowel resection because of the malignancy risk. A patient with anorexia, weight loss, and abdominal pain visited our clinic. A physical exam and imaging study revealed no acute peritoneal signs. A colonoscopy for biopsy and bowel reduction was attempted. The tissue sample was consistent with intestinal tuberculosis. We report intestinal tuberculosis complicating intussusception which was treated without surgical intervention.

Heterogeneous perivascular cell coverage affects breast cancer metastasis and response to chemotherapy.

Angiogenesis and co-optive vascular remodeling are prerequisites of solid tumor growth. Vascular heterogeneity, notably perivascular composition, may play a critical role in determining the rate of cancer progression. The contribution of vascular pericyte heterogeneity to cancer progression and therapy response is unknown. Here, we show that angiopoietin-2 (Ang2) orchestrates pericyte heterogeneity in breast cancer with an effect on metastatic disease and response to chemotherapy. Using multispectral imaging of human breast tumor specimens, we report that perivascular composition, as defined by the ratio of PDGFRß- and desmin+ pericytes, provides information about the response to epirubicin but not paclitaxel. Using 17 distinct patient-derived breast cancer xenografts, we demonstrate a cancer cell-derived influence on stromal Ang2 production and a cancer cell-defined control over tumor vasculature and perivascular heterogeneity. The aggressive features of tumors and their distinct response to therapies may thus emerge by the cancer cell-defined engagement of distinct and heterogeneous angiogenic programs.