MicroRNA-21 induces resistance to the anti-tumour effect of interferon-α/5-fluorouracil in hepatocellular carcinoma cells.

BACKGROUND: We reported recently the clinical efficiency of interferon (IFN)-α/5-fluorouracil (5-FU) combination therapy in advanced hepatocellular carcinoma (HCC). However, prediction of the response to the combination therapy remains unsatisfactory. The aim of this study was to investigate the anti-tumour effects of microRNA (miR)-21 on the sensitivity of HCC cells to IFN-α/5-FU and whether miR-21 can be used as a predictor of the response to such therapy in HCC. METHODS: Changes in the sensitivity of HCC cells (PLC/PRF/5 and HepG2) to IFN-α/5-FU were examined after transfection with pre-miR-21 or anti-miR-21. The correlation between miR-21 expression level, evaluated by qRT-PCR, and response to the therapy was also investigated in clinical HCC specimens. RESULTS: Hepatocellular carcinoma cells transfected with pre-miR-21 were significantly resistant to IFN-α/5-FU. Annexin V assay showed that the percentage of apoptotic cells was significantly lower in cells transfected with pre-miR-21 than control cells. Transfection of anti-miR-21 rendered HCC cells sensitive to IFN-α/5-FU, and such sensitivity was weakened by transfection of siRNAs of target molecules, PETN and PDCD4. miR-21 expression in clinical HCC specimens was significantly associated with the clinical response to the IFN-α/5-FU combination therapy and survival rate. CONCLUSIONS: The miR-21 in HCC cell lines and clinical HCC samples is a significant modulator of the anti-tumour effect of IFN-α and 5-FU. This suggests that miR-21 is a potentially suitable marker for the prediction of the clinical response to the IFN-α/5-FU combination therapy.

Effective application of ET-Kyoto solution for clinical lung transplantation.

The shortage of lung donors and ischemia-reperfusion injury following transplantation have been grave problems in lung transplantation (LTx). One of the most important strategies to solve these problems is the development of effective and highly reliable methods for lung preservation. Therefore, we developed a new organ preservation solution, namely, the extracellular-type trehalose-containing Kyoto (ET-Kyoto) solution. Here we report the first experience of clinical application of ET-Kyoto solution for cadaveric LTx. The recipient was a 38-year-old man with pulmonary emphysema. The donor was a 51-year-old male current smoker with a smoking history of 62 pack-years. The ventilated donor's PaO(2) was 340 Torr (FiO(2) = 1.0). The pulmonary vasculature was flushed with ET-Kyoto solution supplemented with nitroglycerine and dibutyryl cAMP. The recipient underwent bilateral sequential LTx on cardiopulmonary bypass. The ischemic time was 544 and 613 minutes for the left and right lung, respectively. PaO(2) (FiO(2) = 1.0) was 385 Torr immediately after reperfusion. The donor lung was so large that bilateral partial resections were performed at 413 minutes (right) and 348 minutes (left) after reperfusion. On histopathologic examination of the resected transplanted lungs the structure was almost normal. Postoperatively, PaO(2) (FiO(2) = 1.0) was over 400 Torr with or maximum of 526 Torr. The clinical course was almost uneventful. In conclusion, ET-Kyoto solution may be safely applied in clinical cadaveric LTx with extended donor lungs and relatively long ischemic times. Functional and histopathological efficiency of ET-Kyoto solution was confirmed. Longer preservation times with preserved quality using ET-Kyoto solution would increase the donor pool and enable semielective LTx.

Microbial counts, fermentation products, and aerobic stability of whole crop corn and a total mixed ration ensiled with and without inoculation of Lactobacillus casei or Lactobacillus buchneri.

Whole crop corn (DM 29.2%) and a total mixed ration (TMR, DM 56.8%) containing wet brewers grains, alfalfa hay, dried beet pulp, cracked corn, soybean meal, and molasses at a ratio of 5:1:1:1:1:1 on fresh weight basis, were ensiled with and without Lactobacillus casei or Lactobacillus buchneri in laboratory silos. The effects of inoculation on microbial counts, fermentation products, and aerobic stability were determined after 10 and 60 d. Untreated corn silage was well preserved with high lactic acid content, whereas large numbers of remaining yeasts resulted in low stability on exposure to air. Inoculation with L. casei suppressed heterolactic fermentation, but no improvements were found in aerobic stability. The addition of L. buchneri markedly enhanced the aerobic stability, while not affecting the DM loss and NH3-N production. Large amounts of ethanol were found when the TMR was ensiled, and the content of ethanol overwhelmed that of lactic acid in untreated silage. This fermentation was related to high yeast populations and accounted for a large loss of DM found in the initial 10 d. The ethanol production decreased when inoculated with L. casei and L. buchneri, but the effects diminished at 60 d of ensiling. Inoculation with L. buchneri lowered the yeasts in TMR silage from the beginning of storage; however, the populations decreased to undetectable levels when stored for 60 d, regardless of inoculation. No heating was observed in TMR silage during aerobic deterioration test for 7 d. This stability was achieved even when a high population of yeasts remained and was not affected by either inoculation or ensiling period. The results indicate that inoculation with L. buchneri can inhibit yeast growth and improve aerobic stability of corn and TMR silage; however, high stability of TMR silage can be obtained even when no treatments were made and high population (>10(5) cfu/g) of yeasts were detected.

Treatment of intestinal graft-versus-host disease using betamethasone enemas.

Intestinal graft-versus-host disease (GVHD) can readily easily induce generalized metabolic disturbance that influences morbidity and mortality after allogeneic bone marrow transplantation. Although adding a new drug or increasing the doses of immunosuppressive agents will probably be effective for controlling intestinal GVHD, the systemic side effects of such therapy cannot be ignored. In this study, we used betamethasone retention enemas as a local treatment for eight patients with refractory and/or severe intestinal GVHD. Six of the eight patients showed improvement of diarrhea and/or abdominal pain, with a reduction in the stage of GVHD. When treatment with betamethasone enemas was continued for 10 to 27 days in the 6 responding patients, no severe toxicity was observed. One patient failed to respond to treatment and another could not tolerate the enemas. Despite some uncertainty regarding the indications and duration of treatment, betamethasone enemas seem to be a potential alternative method for the management of intestinal GVHD.

Do mammalian cells synthesize lipoic acid? Identification of a mouse cDNA encoding a lipoic acid synthase located in mitochondria.

Lipoic acid is a coenzyme essential to the activity of enzymes such as pyruvate dehydrogenase, which play important roles in central metabolism. However, neither the enzymes responsible for biosynthesis nor the biosynthetic event of lipoic acid has been reported in mammalian cells. In this study, a mouse mLIP1 cDNA for lipoic acid synthase has been identified. We have shown that the cDNA encodes a lipoic acid synthase by its ability to complement a mutant of Escherichia coli defective in lipoic acid synthase and that mLIP1 is targeted into the mitochondria. These findings suggest that mammalian cells are able to synthesize lipoic acid in mitochondria.

Apoptosis and p53 status predict the efficacy of postoperative administration of UFT in non-small cell lung cancer.

To examine whether efficacy of postoperative oral administration of UFT, a 5-fluorouracil derivative chemotherapeutic agent, may be influenced by incidence of apoptosis (apoptosis index) or apoptosis-related gene status (p53 and bcl-2) of the tumour, a total of 162 patients with pathologic stage I non-small cell lung cancer were retrospectively reviewed. UFT was administrated postoperatively to 44 patients (UFT group), and not to the other 118 patients (Control group). For all patients, 5-year survival rate of the UFT group (79.9%) seemed higher than that of the Control group (69.8%), although without significant difference (P = 0.054). For patients with higher apoptotic index, 5-year survival rate of the UFT group (83.3%) was significantly higher than that of the Control group (67.6%, P = 0.039); for patients with lower apoptotic index, however, there was no difference in the prognosis between these two groups. Similarly, UFT was effective for patients without p53 aberrant expression (5-year survival rates: 95.2% for the UFT group and 74.3% for the Control group, P = 0.022), whereas not effective for patients with p53 aberrant expression. Bcl-2 status did not influence the efficacy of UFT. In conclusion, apoptotic index and p53 status are useful factors to predict the efficacy of postoperative adjuvant therapy using UFT.

Molecular cloning and characterization of CHM1L, a novel membrane molecule similar to chondromodulin-I.

Chondromodulin-I (ChM-I) is a cartilage-specific glycoprotein that stimulates the growth of chondrocytes and inhibits the tube formation of endothelial cells. In the present study, we identified a novel ChM-I like molecule, designated ChM1L. Cloning of full length cDNAs of human, mouse, and rat ChM1L revealed that ChM1L encodes 317 amino acids novel type II transmembrane protein. ChM1L protein was expressed on the cell surface as N-glycosylated and non-N-glycosylated protein with molecular mass of 45 and 40 kDa, respectively. In adult mouse tissues, ChM1L mRNA was highly expressed in eye, skeletal muscle, and whole rib. The temporal pattern of ChM1L mRNA was examined using whole embryo at day 10 to 19 of gestation. After day 11, ChM1L mRNA was detected and its level was progressively elevated in association with development of mouse embryo. These data suggest that ChM1L is a novel membrane molecule which is similar to ChM-I that plays a regulatory role in eye, skeletal muscle, and development of embryo.

Requirement of phosphatidylglycerol for photosynthetic function in thylakoid membranes.

To investigate the role of phosphatidylglycerol (PG) in photosynthesis, we constructed a mutant defective in the CDP-diacylglycerol synthase gene from a cyanobacterium, Synechocystis sp. PCC6803. The mutant, designated as SNC1, required PG supplementation for growth. Growth was repressed in PG-free medium concomitantly with the decrease in cellular content of PG. These results indicate that PG is essential, and that SNC1 is defective in PG synthesis. Decrease in PG content was accompanied by a reduction in the cellular content of chlorophyll, but with little effect on the contents of phycobilisome pigments, which showed that levels of chlorophyll-protein complexes decreased without alteration of those of phycobilisomes. Regardless of the decrease in the PG content, CO(2)-dependent photosynthesis by SNC1 was similar to that by the wild type on a chlorophyll basis, but consequently became lower on a cell basis. Simultaneously, the ratio of oxygen evolution of photosystem II (PSII) measured with p-benzoquinone to that of CO(2)-dependent photosynthesis, which ranged between 1.3 and 1.7 in the wild type. However, it was decreased in SNC1 from 1.3 to 0.4 during the early growth phase where chlorophyll content and CO(2)-dependent photosynthesis were little affected, and then finally to 0.1, suggesting that PSII first lost its ability to reduce p-benzoquinone and then decreased in its level and actual activity. These results indicate that PG contributes to the accumulation of chlorophyll-protein complexes in thylakoid membranes, and also to normal functioning of PSII.

[Psychopathology and treatment of traumatic mental disorders--on the vicissitude and controversy around theory and practice].

Recently, among advanced nations, including the United States, there have been growing interest in trauma related psychopathology. Regarding Japan, since Hanshin Awaji Great Earthquake and Sarin gas terrorism by Aum Shinrikyo, interest for psychological trauma has grown high. It is only about twenty years since active research and discussion around these issues started. Therefore, there are still hot dispute around psychopathology of these disorders. Also, therapeutic method is ever progressing. Regarding psychotherapy, there remain a lot of dispute and no general agreement has been made. In this paper, I would like to summarize the literature on psychological trauma and trauma-related psychopathology historically and provide current overview about the theory and practice of this pathology. The summary of the overview is as follows: 1. Psychological trauma is defined as a state caused by traumatic event described in DSM-IV, not as a traumatic event itself. Traumatic mental disorder should be understood as a spectrum of disorders, ranging from phobias and panic reactions related to distinct traumatic events to personality disorders, where intense and chronic traumatic experience is integrated into the personality organization. 2. Historically speaking, traumatic disorders were studied from two directions; war neuroses and etiology of hysteria. Each is corresponds to physioneurosis model and memory processing disturbance model, respectively, which is thought to be two major etiological models of this disorder. 3. Regarding treatment, although corresponding therapeutic idea for each model is provided, currently, most promising treatments are pharmacotherapy using SSRI and novel cognitive-behavioral techniques such as PE (prolonged exposure treatment), SIT (stress inoculation training) and EMDR (eye movement desensitization and reprocessing). As for psychotherapy, hot arguments has been done as to how authentic recovered traumatic memories are and how to deal with them. Regardless of these disputes, it is essential to establish empathic therapeutic bond with the patients.

[Decrease in serum propofol concentrations after acute autologous blood letting].

A change in serum propofol concentrations associated with acute autologous blood letting during anesthesia was investigated in seven scheduled surgical patients. Anesthesia was induced with propofol 2 mg.kg-1 and maintained with infusion of propofol 6 mg.kg-1.hr-1 at a constant rate. After achieving a stable anesthesia, about 10 g.kg-1 of autologous blood was withdrawn in about 15 minutes and 20 ml.kg-1 of acetated Ringer's solution was infused to manage the hypotension caused by withdrawal. A blood sample each 4 ml was taken before and 0, 5, 15, 30 minutes after blood withdrawing. Another 7 patients were anesthetized with the same procedure without blood letting to distinguishing the effect of blood letting from rapid infusion therapy of crystalloid. Assay of serum concentration of propofol was performed with HPLC-spectrofluorometry. Concentrations of propofol were significantly decreased from 2.8 micrograms.ml-1 to 2.3 micrograms.ml-1 just after blood letting, and remained at 2.3 micrograms.ml-1 after 30 minutes from letting. Rapid infusion therapy also decreased the concentrations of propofol from 2.4 micrograms.ml-1 to 1.7 micrograms.ml-1. Continuous infusions of propofol may become a major method of general anesthesia with target controlled infusion techniques (TCI) in clinical settings for the accuracy and reliability of prediction of blood concentrations. However, this study demonstrated unexpected decreases of concentration of propofol during acute autologous blood letting similar to surgical mass bleeding, which might be mainly caused by rapid infusion therapy. The rate of infusion of anesthetic should be readjusted to counteract the effect of acute blood loss or volume replacement.

Significance of cyclic adenosine monophosphate and nitroglycerin in ET-Kyoto solution for lung preservation.

BACKGROUND: We previously demonstrated that the supplement of both dibutyryl cyclic adenosine monophosphate (db-cAMP) and nitroglycerin to the conventional ET-Kyoto solution improved lung preservation significantly. However, the significance of each component in lung preservation remained unclear. We examined the efficacy of the two components on lung preservation in the current study. METHODS: Rat lung grafts (eight per group) were studied in an isolated lung perfusion model. Group 1 grafts were flushed and preserved with ET-Kyoto solution containing 2 mmol/L of db-cAMP. Group 2 grafts were flushed and preserved with ET-Kyoto solution containing 100 mg/L of nitroglycerin. In group 3, the grafts were flushed and preserved with ET-Kyoto solution containing neither db-cAMP nor nitroglycerin as control group. After 4-hour cold storage, the lung grafts were reperfused for 50 minutes. RESULTS: The lung grafts in groups 1 and 2 showed significantly better lung function after reperfusion than those in group 3 with regard to arterial oxygen tension, shunt fraction, peak inspiratory airway pressure, mean pulmonary arterial pressure, and pulmonary vascular resistance. The supplementation of db-cAMP improved especially the pulmonary arterial pressure and pulmonary vascular resistance, while the supplementation of nitroglycerin improved especially the oxygenation and airway pressure of the grafts. CONCLUSIONS: Both of db-cAMP and nitroglycerin had beneficial effects on lung preservation and are essential to the ET-Kyoto solution. There was a difference between the two components in the effects on preserved lungs.

[Problems and strategies in the treatment of mental disorders in elderly patients with physical illness].

There is a high prevalence of mental disorders in the community population of older adults, especially in medical treatment facilities. Therefore, clinicians who treat geriatric patients cannot neglect the psychiatric vulnerability of the elderly population. The fragility of psychological functioning of the elderly is caused not only by psychological contributors, such as various kinds of experiences of loss, but also by biological factors such as decreases in neurotransmitters and in the number of neurons. Another point geriatric clinicians should pay attention to is the powerful mind-body connection in the elderly. Recent psychoneuroimmunological research demonstrates that depression or other types of emotional stress damages the immune system, which can induce some physical diseases. This is especially true for the elderly, who have weakened cell-mediated immune function and are more susceptible to influence by the damaged immune function caused by such psychiatric dysfunction. Also, depression in the elderly can often lead to malnutrition or dehydration, which can induce various kinds of physical illness. On the other hand, physical illness in the elderly can induce depression, because of the psychological vulnerability of the elderly. Due to the strong mind-body connection in the elderly, the availability of psychiatric care is essential. When providing psychiatric care for the elderly, the clinician should attend to all symptoms, not minimizing the importance of biological treatment, while also trying to support the elderly patients psychologically through acceptance of their need for interdependency and respect for their narcissism.

The history, mechanism and clinical use of oral 5-fluorouracil derivative chemotherapeutic agents.

The role of oral chemotherapy has been getting expanded because of the potential advantage in patients' convenience and better quality of life as well as in cost-effectiveness as compared with intravenous chemotherapy. In this article, the history, mechanism of anti-tumor activity, and clinical use of oral chemotherapy using 5-fluorouracil (5-FU) derivative chemotherapeutic agents are reviewed. Pharmacological analysis has revealed that 5-FU, a basic chemotherapeutic agent widely used against a variety of malignant tumors, shows a time dependent anti-tumor activity, and that continuous maintenance of 5-FU concentration in blood is the optimal method in 5-FU administration. UFT, a combination drug of ftorafur (tetrahydrofuranyl-5-fluorouracil, tegafur, FT) and uracil, has been developed to have potent anti-tumor activity by maintaining higher 5-FU concentration in blood and tumor tissues for a long time. FT is a pro-drug that releases 5-FU continuously, and uracil is added to inhibit degradation of the released 5-FU. Clinically, oral administration of UFT has proved to be effective as an adjuvant therapy after surgery for some malignant tumors such as non-small cell lung cancer. Moreover, UFT has proved to be effective for inoperable advanced malignancies such as colorectal cancer, especially in combination with leucovorin or cisplatin. Recently, S-1, a more active oral 5-FU derivative chemotherapeutic agent has been developed in Japan. Several factors to affect anti-tumor effects and/or toxicities of 5-FU and the derivatives, such as thymidylate synthase activity, dehydropyrimidine dehydrogenase activity and p53 status, are also discussed in the article. In conclusion, oral administration of 5-FU derivatives such as UFT may have several clinical advantages over intravenous 5-FU administration.

Clinical application of biodegradable rib connecting pins in thoracotomy.

BACKGROUND: To assess the usefulness in rib reconstruction of biodegradable connecting pins made of polylactide (PLA), PLA rib pins were compared with conventional non-absorbent aluminous ceramic rib pins (alumina rib pins) in terms of their ability for fixing and healing of cut ribs in thoracic surgery. METHODS: There were 13 cases of rib fixation after thoracotomy using PLA rib pins and 11 cases using alumina rib pins, all of which were inserted into the medulla of the stump of the ribs that had been cut at thoracotomy to secure a larger surgical field. The observation period was 6 months after the operation. RESULTS: The degree of vertical shift of the ribs connected with PLA rib pins was significantly reduced compared with the degree using alumina rib pins. In chest radiographs clear zones around the pins, indicating a delay in bone neogenesis, were observed frequently around the alumina rib pins but were absent around the PLA rib pins. Osteosynthesis using PLA rib pins was significantly more favorable than with alumina rib pins. CONCLUSIONS: These results demonstrated that the PLA rib pin was superior to the alumina rib pin for fixing and healing of the cut rib.

L-tryptophan-kynurenine pathway metabolite 3-hydroxyanthranilic acid induces apoptosis in macrophage-derived cells under pathophysiological conditions.

Accumulation of L-kynurenine and 3-hydroxyanthranilic acid (3HAA) occurs in the monocyte-derived cells following immune stimulation, and may derive from L-tryptophan following induction of indoleamine-2,3-dioxygenase. In the present study, we evaluate the possibility that 3HAA acts as an endogenous inducer of monocyte/macrophage apoptosis. Supplementation with 200 microM of 3HAA, but not other L-tryptophan metabolites tested, significantly increased the number of apoptotic cells in both THP-1 and U937 cells. Catalase, superoxide dismutase and manganese ions markedly enhanced apoptosis in the presence of 3HAA in these cells. The present results suggest that 3HAA induces the macrophage/monocyte apoptosis under certain conditions, which may be relevant to pathophysiology of inflammatory conditions.

Biosynthesis of lipoic acid in Arabidopsis: cloning and characterization of the cDNA for lipoic acid synthase.

Lipoic acid is a coenzyme that is essential for the activity of enzyme complexes such as those of pyruvate dehydrogenase and glycine decarboxylase. We report here the isolation and characterization of LIP1 cDNA for lipoic acid synthase of Arabidopsis. The Arabidopsis LIP1 cDNA was isolated using an expressed sequence tag homologous to the lipoic acid synthase of Escherichia coli. This cDNA was shown to code for Arabidopsis lipoic acid synthase by its ability to complement a lipA mutant of E. coli defective in lipoic acid synthase. DNA-sequence analysis of the LIP1 cDNA revealed an open reading frame predicting a protein of 374 amino acids. Comparisons of the deduced amino acid sequence with those of E. coli and yeast lipoic acid synthase homologs showed a high degree of sequence similarity and the presence of a leader sequence presumably required for import into the mitochondria. Southern-hybridization analysis suggested that LIP1 is a single-copy gene in Arabidopsis. Western analysis with an antibody against lipoic acid synthase demonstrated that this enzyme is located in the mitochondrial compartment in Arabidopsis cells as a 43-kD polypeptide.

Evaluation of new improved solution containing trehalose in free skin flap storage.

BACKGROUND DATA: In this study, we evaluated a new intracellular type (IT-K) solution containing trehalose in a rabbit free skin flap storage model. Trehalose is a nonreducing disaccharide that can stabilise cell membranes under various stressful conditions. MATERIAL AND METHOD: Seventy-two free skin flaps of the ear of rabbits were preserved in Euro-Collins (EC) solution or in IT-K solution for 24, 48, and 72 h at 4 degrees C. After completion of preservation, these flaps were replanted to the other ear by microsurgical techniques. Viability study and photo documentation were performed daily for 7 days. Tissue specimens were taken 24 h after vascular anastomosis, fixed in 10% formaldehyde and stained with haematoxylin and eosin (HE). Survival rates were analysed by Fisher's exact test for comparison of the two experimental groups. Values of P < 0.05 were considered to be statistically significant. RESULTS: After 7 days, a survival rate of 100% of flaps were observed in both solutions after 24 h of preservation. After preservation for 48 h in IT-K solution the survival rate was 100%. However, in EC solution survival decreased to 75% (9 of 12 preserved flaps survived). This difference increased to 33.3% (4 of 12 flaps) in EC solution and 91.6% (11 of 12 flaps) (P < 0.01) in IT-K solution when the flaps were stored for 72 h. Light microscopic examination also showed less damage in flaps preserved in IT-K solution than in these preserved in EC solution. CONCLUSION: IT-K solution was superior to EC solution in the preservation of free skin flaps on rabbit ears when stored for 48 and 72 h.

Partial purification and characterization of a gibberellin-binding protein from seedlings of Azukia angularis.

Little is known about mechanisms for the perception of gibberellin (GA), a phytohormone, by plants. A GA-binding protein (GBP) with receptor-like characteristics was partially purified from azuki bean (Azukia angularis) seedlings. Gibberellins that caused much elongation of azuki epicotyl sections showed strong binding affinity for the GBP, and their biosynthetic precursors and inactive metabolites had weak affinity for it. The apparent Kd value for GA4 was calculated to be 7 x 10(-10) M from Scatchard plots. The molecular weight of the GBP in the native state was estimated to be 25,000 by high-performance liquid chromatography with a gel-permeation column. The GBP may be one of the receptor candidates for GAs.

[Benign infantile mitochondrial myopathy caused by reversible cytochrome c oxidase deficiency].

A 2-month-old girl had generalized weakness, profound muscular hypotonia, hepatomegaly and severe lactic acidosis. She needed ventilatory support. Muscle specimen taken at 2 months showed ragged-red fibers, abnormal mitochondria, and reduced cytochrome c oxidase (CCO) staining Biochemical analysis showed CCO activity to be reduced to about 16% of the normal mean. She received carnitine and coenzyme Q10 supplementation from the age of 3 months and abnormal blood lactate values declined to near normal values during the first three weeks. Gradually her condition started to improved: she held her head at 9 months, and walked alone at 15 months. The second biopsy specimen at 3 years and 8 months showed almost normal CCO staining and she was free of clinical signs. This case is an example of a rare benign infantile mitochondrial myopathy caused by CCO deficiency. Early diagnosis is crucial to provide intensive treatment until spontaneous clinical improvement appears. We concluded that carnitine and coenzyme Q10 supplementation was a useful treatment for clinical improvement in patients with a benign infantile mitochondrial myopathy caused by CCO deficiency.