Ketone Body Signaling Mediates Intestinal Stem Cell Homeostasis and Adaptation to Diet.

Little is known about how metabolites couple tissue-specific stem cell function with physiology. Here we show that, in the mammalian small intestine, the expression of Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthetase 2), the gene encoding the rate-limiting enzyme in the production of ketone bodies, including beta-hydroxybutyrate (ßOHB), distinguishes self-renewing Lgr5+ stem cells (ISCs) from differentiated cell types. Hmgcs2 loss depletes ßOHB levels in Lgr5+ ISCs and skews their differentiation toward secretory cell fates, which can be rescued by exogenous ßOHB and class I histone deacetylase (HDAC) inhibitor treatment. Mechanistically, ßOHB acts by inhibiting HDACs to reinforce Notch signaling, instructing ISC self-renewal and lineage decisions. Notably, although a high-fat ketogenic diet elevates ISC function and post-injury regeneration through ßOHB-mediated Notch signaling, a glucose-supplemented diet has the opposite effects. These findings reveal how control of ßOHB-activated signaling in ISCs by diet helps to fine-tune stem cell adaptation in homeostasis and injury.

SOX17 enables immune evasion of early colorectal adenomas and cancers.

A hallmark of cancer is the avoidance of immune destruction. This process has been primarily investigated in locally advanced or metastatic cancer1-3; however, much less is known about how pre-malignant or early invasive tumours evade immune detection. Here, to understand this process in early colorectal cancers (CRCs), we investigated how naive colon cancer organoids that were engineered in vitro to harbour Apc-null, KrasG12D and Trp53-null (AKP) mutations adapted to the in vivo native colonic environment. Comprehensive transcriptomic and chromatin analyses revealed that the endoderm-specifying transcription factor SOX17 became strongly upregulated in vivo. Notably, whereas SOX17 loss did not affect AKP organoid propagation in vitro, its loss markedly reduced the ability of AKP tumours to persist in vivo. The small fraction of SOX17-null tumours that grew displayed notable interferon-γ (IFNγ)-producing effector-like CD8+ T cell infiltrates in contrast to the immune-suppressive microenvironment in wild-type counterparts. Mechanistically, in both endogenous Apc-null pre-malignant adenomas and transplanted organoid-derived AKP CRCs, SOX17 suppresses the ability of tumour cells to sense and respond to IFNγ, preventing anti-tumour T cell responses. Finally, SOX17 engages a fetal intestinal programme that drives differentiation away from LGR5+ tumour cells to produce immune-evasive LGR5- tumour cells with lower expression of major histocompatibility complex class I (MHC-I). We propose that SOX17 is a transcription factor that is engaged during the early steps of colon cancer to orchestrate an immune-evasive programme that permits CRC initiation and progression.

Short-term post-fast refeeding enhances intestinal stemness via polyamines.

For over a century, fasting regimens have improved health, lifespan and tissue regeneration in diverse organisms, including humans1-6. However, how fasting and post-fast refeeding affect adult stem cells and tumour formation has yet to be explored in depth. Here we demonstrate that post-fast refeeding increases intestinal stem cell (ISC) proliferation and tumour formation; post-fast refeeding augments the regenerative capacity of Lgr5+ ISCs, and loss of the tumour suppressor gene Apc in post-fast-refed ISCs leads to a higher tumour incidence in the small intestine and colon than in the fasted or ad libitum-fed states, demonstrating that post-fast refeeding is a distinct state. Mechanistically, we discovered that robust mTORC1 induction in post-fast-refed ISCs increases protein synthesis via polyamine metabolism to drive these changes, as inhibition of mTORC1, polyamine metabolite production or protein synthesis abrogates the regenerative or tumorigenic effects of post-fast refeeding. Given our findings, fast-refeeding cycles must be carefully considered and tested when planning diet-based strategies for regeneration without increasing cancer risk, as post-fast refeeding leads to a burst in stem-cell-driven regeneration and tumorigenicity.

Stiffness Restricts the Stemness of the Intestinal Stem Cells and Skews Their Differentiation Toward Goblet Cells.

BACKGROUND & AIMS: Fibrosis and tissue stiffening are hallmarks of inflammatory bowel disease (IBD). We have hypothesized that the increased stiffness directly contributes to the dysregulation of the epithelial cell homeostasis in IBD. Here, we aim to determine the impact of tissue stiffening on the fate and function of the intestinal stem cells (ISCs). METHODS: We developed a long-term culture system consisting of 2.5-dimensional intestinal organoids grown on a hydrogel matrix with tunable stiffness. Single-cell RNA sequencing provided stiffness-regulated transcriptional signatures of the ISCs and their differentiated progeny. YAP-knockout and YAP-overexpression mice were used to manipulate YAP expression. In addition, we analyzed colon samples from murine colitis models and human IBD samples to assess the impact of stiffness on ISCs in vivo. RESULTS: We demonstrated that increasing the stiffness potently reduced the population of LGR5+ ISCs and KI-67+-proliferating cells. Conversely, cells expressing the stem cell marker, olfactomedin-4, became dominant in the crypt-like compartments and pervaded the villus-like regions. Concomitantly, stiffening prompted the ISCs to preferentially differentiate toward goblet cells. Mechanistically, stiffening increased the expression of cytosolic YAP, driving the extension of olfactomedin-4+ cells into the villus-like regions, while it induced the nuclear translocation of YAP, leading to preferential differentiation of ISCs toward goblet cells. Furthermore, analysis of colon samples from murine colitis models and patients with IBD demonstrated cellular and molecular remodeling reminiscent of those observed in vitro. CONCLUSIONS: Collectively, our findings highlight that matrix stiffness potently regulates the stemness of ISCs and their differentiation trajectory, supporting the hypothesis that fibrosis-induced gut stiffening plays a direct role in epithelial remodeling in IBD.

Role of Csk in intestinal epithelial barrier function and protection against colitis.

Intestinal epithelial cells (IECs) play a pivotal role in the maintenance of the integrity and barrier function of the intestinal epithelium. Dysfunctions of IECs are thought to participate in the disruption of the intestinal epithelial barrier, resulting in gastrointestinal diseases, such as colitis and colorectal cancer. Here we show that IEC-specific COOH-terminal Src kinase (Csk)-deficient mice (Csk CKO mice) manifested the increased susceptibility to dextran sodium sulfate (DSS)-induced colitis, a model of inflammatory bowel disease. DSS-treated Csk CKO mice also exhibited the significantly elevated intestinal permeability. Following DSS treatment, Csk CKO mice exhibited the higher proliferative activity of colonic epithelial cells and the increased number of apoptotic cells in the colon compared with that apparent for control mice. Moreover, the abundance of the tight junction protein occludin, which regulates cell-cell adhesion as well as epithelial permeability, was markedly reduced in the colon of DSS-treated Csk CKO mice. These results thus suggest that Csk in IECs plays important roles in the regulation of the intestinal epithelial barrier function and protection against colitis.

Protein tyrosine phosphatase SAP-1 protects against colitis through regulation of CEACAM20 in the intestinal epithelium.

Intestinal epithelial cells contribute to regulation of intestinal immunity in mammals, but the detailed molecular mechanisms of such regulation have remained largely unknown. Stomach-cancer-associated protein tyrosine phosphatase 1 (SAP-1, also known as PTPRH) is a receptor-type protein tyrosine phosphatase that is localized specifically at microvilli of the brush border in gastrointestinal epithelial cells. Here we show that SAP-1 ablation in interleukin (IL)-10-deficient mice, a model of inflammatory bowel disease, resulted in a marked increase in the severity of colitis in association with up-regulation of mRNAs for various cytokines and chemokines in the colon. Tyrosine phosphorylation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20, an intestinal microvillus-specific transmembrane protein of the Ig superfamily, was greatly increased in the intestinal epithelium of the SAP-1-deficient animals, suggesting that this protein is a substrate for SAP-1. Tyrosine phosphorylation of CEACAM20 by the protein tyrosine kinase c-Src and the consequent association of CEACAM20 with spleen tyrosine kinase (Syk) promoted the production of IL-8 in cultured cells through the activation of nuclear factor-κB (NF-κB). In addition, SAP-1 and CEACAM20 were found to form a complex through interaction of their ectodomains. SAP-1 and CEACAM20 thus constitute a regulatory system through which the intestinal epithelium contributes to intestinal immunity.

Pretreatment with magnesium sulfate attenuates white matter damage by preventing cell death of developing oligodendrocytes.

AIM: Antenatal maternal administration of magnesium sulfate (MgSO4 ) reduces cerebral palsy in preterm infants. However, it remains controversial as to whether it also reduces occurrence of white matter damage, or periventricular leukomalacia. We assessed the effect of MgSO4 against white matter damage induced by hypoxic-ischemic insult using a neonatal rat model and culture of premyelinating oligodendrocytes (pre-OL). METHODS: Rat pups at postnatal day (P) 6 were administered either MgSO4 or vehicle intraperitoneally before hypoxic-ischemic insult (unilateral ligation of the carotid artery followed by 6% oxygen for 1 h). The population of oligodendrocyte (OL) markers and CD-68-positive microglia at P11, and TdT-mediated biotin-16-dUTP nick-end labeling (TUNEL)-positive cells at P8 were evaluated in pericallosal white matter. Primary cultures of mouse pre-OL were subjected to oxygen glucose deprivation condition, and the lactate dehydrogenase release from culture cells was evaluated to assess cell viability. RESULTS: Pretreatment with MgSO4 attenuated the loss of OL markers, such as myelin basic protein and Olig2, in ipsilateral pericallosal white matter and decreased the number of CD-68-positive microglia and TUNEL-positive cells in vivo. Pretreatment with MgSO4 also inhibited lactate dehydrogenase release from pre-OL induced by oxygen glucose deprivation in vitro. CONCLUSION: Pretreatment with MgSO4 attenuates white matter damage by preventing cell death of pre-OL.

Regulation by gut commensal bacteria of carcinoembryonic antigen-related cell adhesion molecule expression in the intestinal epithelium.

Carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 1 and CEACAM20, immunoglobulin superfamily members, are predominantly expressed in intestinal epithelial cells (IECs) and co-localized at the apical surface of these cells. We here showed that the expression of mouse CEACAM1 and CEACAM20 at both mRNA and protein levels was markedly reduced in IECs of the small intestine by the treatment of mice with antibiotics against Gram-positive bacteria. The expression of both proteins was also decreased in IECs of the small intestine from germ-free mice, compared with that from control specific-pathogen-free mice. Exposure of intestinal organoids to IFN-γ markedly increased the expression of either CEACAM1 or CEACAM20, whereas the exposure to TNF-α increased the expression of the former protein, but not that of the latter. In contrast, the expression of CEACAM20, but not of CEACAM1, in intestinal organoids was markedly increased by exposure to butyrate, a short-chain fatty acid produced by bacterial fermentation in the intestine. Collectively, our results suggest that Gram-positive bacteria promote the mRNA expression of CEACAM1 or CEACAM20 in the small intestine. Inflammatory cytokines or butyrate likely participates in such effects of commensal bacteria.

Hypothermia attenuates apoptosis and protects contact between myelin basic protein-expressing oligodendroglial-lineage cells and neurons against hypoxia-ischemia.

Periventricular leukomalacia (PVL) is a major form of brain injury among preterm infants, which is characterized by extensive loss and dysfunction of premyelinating oligodendrocytes (pre-OLs) induced by hypoxia-ischemia (HI). Therapeutic hypothermia, which is a standard treatment for term infants with HI encephalopathy, is not indicated for preterm infants because its safety and effect have not been established. Here we investigate the effectiveness and mechanism of hypothermia for the inhibition of pre-OLs damage in PVL. For in vivo studies, 6-day-old rats underwent left carotid artery ligation, followed by exposure to 6% oxygen for 1 hr under hypothermic or normothermic conditions. The loss of myelin basic protein (MBP) was inhibited by hypothermia. For in vitro studies, primary pre-OLs cultures were subjected to oxygen-glucose deprivation (OGD) under normothermic or hypothermic conditions, and dorsal root ganglion neurons were subsequently added. Hypothermia inhibited apoptosis of pre-OLs, and, despite specific downregulation of 21.5- and 17-kDa MBP mRNA expression during hypothermia, recovery of the expression after OGD was superior compared with normothermia. OGD caused disarrangement of MBP distribution, decreased the levels of phosphorylated 21.5-kDa MBP, and disturbed the capacity to contact with neurons, all of which were restored by hypothermia. Pharmacological inhibition of ERK1/2 phosphorylation with U0126 during and after OGD significantly reduced the protective effects of hypothermia on apoptosis and myelination, respectively. These data suggest that phosphorylated exon 2-containing (21.5- and possibly 17-kDa) MBP isoforms may play critical roles in myelination and that hypothermia attenuates apoptosis and preserves the contact between OLs and neurons via ERK1/2 phosphorylation.

A genetically encoded fluorescent reporter for polyamines.

Polyamines are abundant and evolutionarily conserved metabolites that are essential for life. Dietary polyamine supplementation extends life-span and health-span. Dysregulation of polyamine homeostasis is linked to Parkinson's disease and cancer, driving interest in therapeutically targeting this pathway. However, measuring cellular polyamine levels, which vary across cell types and states, remains challenging. We introduce a first-in-class genetically encoded polyamine reporter for real-time measurement of polyamine concentrations in single living cells. This reporter utilizes the polyamine-responsive ribosomal frameshift motif from the OAZ1 gene. We demonstrate broad applicability of this approach and reveal dynamic changes in polyamine levels in response to genetic and pharmacological perturbations. Using this reporter, we conducted a genome-wide CRISPR screen and uncovered an unexpected link between mitochondrial respiration and polyamine import, which are both risk factors for genetic Parkinson's disease. By offering a new lens to examine polyamine biology, this reporter may advance our understanding of these ubiquitous metabolites and accelerate therapy development.

Inferior vena cava tumor thrombus that directly infiltrated from paracaval lymph node metastases in a patient with recurrent hepatocellular carcinoma.

Herein, we present the case of a patient with recurrent hepatocellular carcinoma (HCC) who had paracaval lymph node (LN) metastases with an inferior vena cava (IVC) tumor thrombus after a hepatectomy. A 65-year-old man with chronic hepatitis B virus infection received an extended anterior segmentectomy because of two hepatic tumors, located in segments 7 and 8. Histological examination of both resected specimens showed mostly moderately differentiated HCC with some poorly differentiated areas, and liver cirrhosis (A2/F4). Because the patient had an elevated α-fetoprotein serum level, abdominal computed tomography (CT) was performed. Abdominal CT revealed a 9-mm-diameter recurrent tumor in hepatic segment 3 and paracaval LN metastases with an IVC tumor thrombus at 8 months after the first operation. The patient received transcatheter arterial chemoembolization as treatment for the intrahepatic recurrence, following resection of the paracaval LN metastases and removal of the IVC tumor thrombus. In this case, the paracaval LN metastases had directly infiltrated the IVC via the lumbar veins, resulting in an IVC tumor thrombus, which usually develops from an intrahepatic tumor via the hepatic vein. The development of an IVC tumor thrombus with HCC recurrence, as in this case, is very rare, and based on a PubMed search, we believe this report may be the first to describe this condition.

Case of isolated lateral lymph node recurrence occurring after TME for T1 lower rectal cancer treated with lateral lymph node dissection: report of a case.

We experienced a rare case of isolated lateral lymph node (LLN) recurrence after laparoscopic intersphincteric resection with total mesorectal excision for T1 lower rectal cancer that was successfully treated using LLN dissection with en bloc resection of the left superior vesical artery, the left ovary and the left obturator nerve, artery and vein. There are no guidelines for treating patients with isolated LLN recurrence because isolated LLN recurrence is rare, especially in patients with T1 rectal cancer. However, in patients with pT1N0 lower rectal cancer, follow-up examinations of the pelvis should be performed periodically. Patients with isolated LLN recurrence treated with surgery might have good long-term prognoses. Therefore, for such patients, surgical resection should be considered as a curative treatment only.

Recurrence of hepatocellular carcinoma presenting as an asymptomatic appendiceal tumor: report of a case.

This report presents the case of a 66-year-old male with appendicular metastasis from hepatocellular carcinoma. He had a clinical history of right lobectomy of the liver after the diagnosis of hepatocellular carcinoma 3 years prior, and was admitted because of an asymptomatic appendiceal tumor detected by computed tomography. The appendiceal tumor was preoperatively suspected to be a recurrence of hepatocellular carcinoma, because of the patient's elevated level of serum α-fetoprotein and protein induced by vitamin K absence or antagonist II, and based on the magnetic resonance imaging findings. An appendectomy was thus performed, and the histopathological findings confirmed the diagnosis of a metastatic tumor from hepatocellular carcinoma. Appendicular metastasis is extremely rare. This is only the second case of a metastatic appendiceal tumor from hepatocellular carcinoma reported in the English literature.

Post-fast refeeding enhances intestinal stem cell-mediated regeneration and tumourigenesis through mTORC1-dependent polyamine synthesis.

For more than a century, fasting regimens have improved health, lifespan, and tissue regeneration in diverse organisms, including humans. However, how fasting and post-fast refeeding impact adult stem cells and tumour formation has yet to be explored in depth. Here, we demonstrate that post-fast refeeding increases intestinal stem cell (ISC) proliferation and tumour formation: Post-fast refeeding augments the regenerative capacity of Lgr5+ intestinal stem cells (ISCs), and loss of the tumour suppressor Apc in ISCs under post-fast refeeding leads to a higher tumour incidence in the small intestine and colon than in the fasted or ad libitum (AL) fed states. This demonstrates that post-fast refeeding is a distinct state. Mechanistically, we discovered that robust induction of mTORC1 in post-fast-refed ISCs increases protein synthesis via polyamine metabolism to drive these changes, as inhibition of mTORC1, polyamine metabolite production, or protein synthesis abrogates the regenerative or tumourigenic effects of post-fast refeeding. Thus, fast-refeeding cycles must be carefully considered when planning diet-based strategies for regeneration without increasing cancer risk, as post-fast refeeding leads to a burst not only in stem cell-driven regeneration but also in tumourigenicity.

Inulin diet uncovers complex diet-microbiota-immune cell interactions remodeling the gut epithelium.

BACKGROUND: The continuous proliferation of intestinal stem cells followed by their tightly regulated differentiation to epithelial cells is essential for the maintenance of the gut epithelial barrier and its functions. How these processes are tuned by diet and gut microbiome is an important, but poorly understood question. Dietary soluble fibers, such as inulin, are known for their ability to impact the gut bacterial community and gut epithelium, and their consumption has been usually associated with health improvement in mice and humans. In this study, we tested the hypothesis that inulin consumption modifies the composition of colonic bacteria and this impacts intestinal stem cells functions, thus affecting the epithelial structure. METHODS: Mice were fed with a diet containing 5% of the insoluble fiber cellulose or the same diet enriched with an additional 10% of inulin. Using a combination of histochemistry, host cell transcriptomics, 16S microbiome analysis, germ-free, gnotobiotic, and genetically modified mouse models, we analyzed the impact of inulin intake on the colonic epithelium, intestinal bacteria, and the local immune compartment. RESULTS: We show that the consumption of inulin diet alters the colon epithelium by increasing the proliferation of intestinal stem cells, leading to deeper crypts and longer colons. This effect was dependent on the inulin-altered gut microbiota, as no modulations were observed in animals deprived of microbiota, nor in mice fed cellulose-enriched diets. We also describe the pivotal role of γδ T lymphocytes and IL-22 in this microenvironment, as the inulin diet failed to induce epithelium remodeling in mice lacking this T cell population or cytokine, highlighting their importance in the diet-microbiota-epithelium-immune system crosstalk. CONCLUSION: This study indicates that the intake of inulin affects the activity of intestinal stem cells and drives a homeostatic remodeling of the colon epithelium, an effect that requires the gut microbiota, γδ T cells, and the presence of IL-22. Our study indicates complex cross kingdom and cross cell type interactions involved in the adaptation of the colon epithelium to the luminal environment in steady state. Video Abstract.

Brain metastasis from colorectal cancer: prognostic factors and survival.

BACKGROUND: Colorectal cancer (CRC) rarely metastasizes to the brain, and the incidence rate has been reported to be 1-2%. Unfortunately, the median survival for patients with brain metastasis (BM) from CRC is short. In this study, we retrospectively investigated the BM from CRC and examined the prognostic factors. METHODS: We retrospectively analyzed 29 CRC patients who developed BM; the lesions were diagnosed synchronously in 1 patient and metachronously in 28 patients. RESULTS: After BM, the median survival time was 7.4 months. In the groups of patients who underwent surgical resection and radiation therapy, the median survival times were 8.3 and 7.4 months, respectively. The difference between the two groups was not statistically significant. The curability of the therapy for BM, number of BM, number of metastatic organs including the brain, and the CEA level at the time of treatment of the BM were significantly associated with the cancer-specific survival (P = 0.0044, 0.0229, 0.0019, and 0.0205, respectively). CONCLUSIONS: The prognosis of patients with BM from CRC was associated with the curability of the therapy for BM, number of metastatic organs, and the serum CEA level. The modality of treatment had no significant impact on the outcome.

Birthweight placental weight ratio of appropriate-for-dates and light-for-dates infants in preterm delivery.

AIM: Although birthweight placental weight ratio (BPR) may be a promising indicator which reflects pathophysiology of fetal growth restriction (FGR), the standard of BPR changes throughout gestation in a Japanese population has not been established as far as we know. Therefore, we first examined BPR of appropriate-for-dates (AFD) infants in each gestational week in preterm deliveries. We then compared it with that in a group of light-for-dates (LFD) infants born from mothers with and without pregnancy-induced hypertension (PIH). MATERIAL AND METHODS: Placentas of a singleton pregnancy with 373 AFD and 110 LFD infants delivered from 22 to 36weeks of gestation in our hospital during the period between September 2000 and December 2008 were included. We examined the placental weight and BPR of each gestational week in AFD and LFD groups. And the mean BPR and placental weight in the three groups (AFD: LFD with PIH: LFD without PIH) were compared according to gestational periods. RESULTS: The placental weight and BPR were significantly correlated to the gestational week both in AFD and LFD groups. We found that although the mean BPR in LFD-PIH(-) group was significantly lower than those both in AFD group and in LFD-PIH(+) group in 22-29weeks, the mean BPR in 30-36weeks was not statistically different among these three groups. CONCLUSION: Our result in the AFD group may be useful as one of the standards of BPR changes throughout gestation in a Japanese population for future studies. We believe that BPR may be a clinically useful indicator which reflects pathophysiology of FGR.

Prenatal diagnosis and management of congenital chloride diarrhea: A case report of 2 siblings.

Congenital chloride diarrhea (CLD) is a rare hereditary disease. The basic defect of CLD is massive loss of Cl(-) and fluid into the ileum and colon. Prenatal diagnosis of this disease is quite important because the infant requires electrolyte supplementation from the early postnatal period. Two cases in which prenatal diagnoses of CLD were made in siblings are reported. Extreme electrolyte imbalance may cause fetal cardiac dysfunction or a poor general condition leading to a non-reassuring fetal status in cases with CLD. Therefore, frequent fetal monitoring using cardiotocograms and ultrasound may be beneficial to some fetuses with CLD to detect fetal deterioration. In addition, repeated amnioreduction may be required to treat severe polyhydramnios and threatened preterm delivery.

Transplacental fetal therapy for junctional ectopic tachycardia.

Junctional ectopic tachycardia (JET) is a rare type of tachyarrhythmia. A 39-year-old woman was transferred to our hospital for fetal tachyarrhythmia at 32 weeks' gestation. Fetal cardiac sonography revealed atrial and ventricular rates of 120-130 and 175-230 bpm, respectively, without 1:1 atrioventricular relationship. As ventricular tachycardia was considered to be the most probable diagnosis, transplacental therapy with digoxin and sotalol was done with partial response. Diagnosis of JET was made after birth. Although fetal JET is rare and prenatal diagnosis and treatment of this condition is still a challenge, differential diagnosis of fetal tachyarrhythmia should include this disorder.

A case of pyruvate dehydrogenase E1α subunit deficiency with antenatal brain dysgenesis demonstrated by prenatal sonography and magnetic resonance imaging.

Prenatal depiction of brain dysgenesis in patients with pyruvate dehydrogenase complex (PDHc) deficiencies has been infrequently reported. As PDHc plays a critical role in the brain that obtains all of the energy from the aerobic oxidation of glucose, its deficiency is a severe inborn disorder of metabolism, which predominantly affects the nervous system. This report describes a case of PDHc deficiency with antenatal brain dysgenesis depicted in detail by fetal ultrasound and magnetic resonance imaging. This is the first case report clearly demonstrating the developing mechanism and time course of antenatal brain lesions in a patient with PDHc deficiency.