Exploring applications of blockchain in healthcare: road map and future directions.

Blockchain technology includes numerous elements such as distributed ledgers, decentralization, authenticity, privacy, and immutability. It has progressed past the hype to find actual use cases in industries like healthcare. Blockchain is an emerging area that relies on a consensus algorithm and the idea of a digitally distributed ledger to eliminate any intermediary risks. By enabling them to trace data provenance and any changes made, blockchain technology can enable different healthcare stakeholders to share access to their networks without violating data security and integrity. The healthcare industry faces challenges like fragmented data, security and privacy concerns, and interoperability issues. Blockchain technology offers potential solutions by ensuring secure, tamper-proof storage across multiple network nodes, improving interoperability and patient privacy. Encrypting patient data further enhances security and reduces unauthorized access concerns. Blockchain technology, deployed over the Internet, can potentially use the current healthcare data by using a patient-centric approach and removing the intermediaries. This paper discusses the effective utilization of blockchain technology in the healthcare industry. In contrast to other applications, the exoteric evaluation in this paper shows that the innovative technology called blockchain technology has a major role to play in the existing and future applications of the healthcare industry and has significant benefits.

Applications of cognitive internet of medical things in modern healthcare.

The sudden outbreak of the novel coronavirus disease in 2019, known as COVID-19 has impacted the entire globe and has forced governments of various countries to a partial or full lockdown in the fear of the rapid spread of this disease. The major lesson learned from this pandemic is that there is a need to implement a robust system by using non-pharmaceutical interventions for the prevention and control of new contagious viruses. This goal can be achieved using the platform of the Internet of Things (IoT) because of its seamless connectivity and ubiquitous sensing ability. This technology-enabled healthcare sector is helpful to monitor COVID-19 patients properly by adopting an interconnected network. IoT is useful for improving patient satisfaction by reducing the rate of readmission in the hospital. The presented work discusses the applications and technologies of IoT like smart and wearable devices, drones, and robots which are used in healthcare systems to tackle the Coronavirus pandemic This paper focuses on applications of cognitive radio-based IoT for medical applications, which is referred to as "Cognitive Internet of Medical Things" (CIoMT). CIoMT is a disruptive and promising technology for dynamic monitoring, tracking, rapid diagnosis, and control of pandemics and to stop the spread of the virus. This paper explores the role of the CIoMT in the health domain, especially during pandemics, and also discusses the associated challenges and research directions.

A systematic review on AI/ML approaches against COVID-19 outbreak.

A pandemic disease, COVID-19, has caused trouble worldwide by infecting millions of people. The studies that apply artificial intelligence (AI) and machine learning (ML) methods for various purposes against the COVID-19 outbreak have increased because of their significant advantages. Although AI/ML applications provide satisfactory solutions to COVID-19 disease, these solutions can have a wide diversity. This increase in the number of AI/ML studies and diversity in solutions can confuse deciding which AI/ML technique is suitable for which COVID-19 purposes. Because there is no comprehensive review study, this study systematically analyzes and summarizes related studies. A research methodology has been proposed to conduct the systematic literature review for framing the research questions, searching criteria and relevant data extraction. Finally, 264 studies were taken into account after following inclusion and exclusion criteria. This research can be regarded as a key element for epidemic and transmission prediction, diagnosis and detection, and drug/vaccine development. Six research questions are explored with 50 AI/ML approaches in COVID-19, 8 AI/ML methods for patient outcome prediction, 14 AI/ML techniques in disease predictions, along with five AI/ML methods for risk assessment of COVID-19. It also covers AI/ML method in drug development, vaccines for COVID-19, models in COVID-19, datasets and their usage and dataset applications with AI/ML.

Effects of Varying Levels of Fungal (Arachniotus sp.) Treated Wheat Straw as an Ingredient of Total Mixed Ration on Growth Performance and Nutrient Digestibility in Nili Ravi Buffalo Calves.

The study was carried out to explore the effects of replacing wheat straw with fungal treated wheat straw as an ingredient of total mixed ration (TMR) on the growth performance and nutrient digestibility in Nili Ravi buffalo male calves. Fungal treated wheat straw was prepared using Arachniotus sp. Four TMRs were formulated where wheat straw was replaced with 0 (TMR1), 33 (TMR2), 67 (TMR3), and 100% (TMR4) fungal treated wheat straw in TMR. All TMRs were iso-caloric and iso-nitrogenous. The experimental TMRs were randomly assigned to four groups of male calves (n = 6) according to completely randomized design and the experiment continued for four months. The calves fed TMR2 exhibited a significant improve in dry matter intake, average daily weight gain, feed conversion ratio and feed economics compared to other groups. The same group also showed higher digestibility of dry matter, crude protein, neutral-, and acid detergent fibers than those fed on other TMRs. It is concluded that TMR with 33% fungal-treated wheat straw replacement has a potential to give an enhanced growth performance and nutrient digestibility in male Nili Ravi buffalo calves.

Influence of weaning regimen on intake, growth characteristics and plasma blood metabolites in male buffalo calves.

Experiment was conducted to evaluate the effect of weaning age on growth performance, feed intake, feed efficiency (FE) and blood metabolites in Nili-Ravi male buffalo (Bubalus bubalis) calves. Twenty-four male buffalo calves were assigned to one of the three treatment groups: continuous milk feeding (CMF), limited milk feeding (LMF) and early weaning (EW), and weaned off milk at 12, 10 and 8 weeks of age, respectively. For the first 3 days after birth, calves in all three treatments were fed colostrum, and were then moved to individual milk feeding at 10% of BW for the next 6 weeks. Thereafter, the provision of milk to the CMF group was gradually tapered to zero through week 12, using week 6 intakes as a base. The LMF calves were fed milk at 7.5%, 5.0%, 3.5%, and 1.5% of BW during weeks 7 to 10, respectively. Lastly, calves in the EW group were fed milk at 5.0% and 2.5% of BW at weeks 7 and 8, respectively. Calf starter (CS) feed was also provided ad libitum from weeks 2 to 12 and individual intakes were recorded on a daily basis. Blood samples were taken from weeks 6 to 12, on a weekly basis; whereas, the BW, heart girth, withers height and hip width were measured at the start of experiment and later on a weekly basis. Weight gain, average daily gain, and body measurements were the same across all three groups. Milk intake was lower (P < 0.05), whereas CS intake was greater (P < 0.05) in the EW calves compared with the other treatment groups. Dry matter intake was greater (P < 0.05) in the EW and LMF calves compared with the CMF calves. The FE was greater (P < 0.05) in the CMF calves compared with the LMF and EW treatment groups. Blood glucose concentration was similar among the treatments; however, blood urea nitrogen was greater (P < 0.05) in the EW calves compared with the CMF and LMF groups. Plasma concentration of non-esterified fatty acids was higher (P < 0.05) in the EW calves compared with the CMF calves. In light of these results, it is evident that buffalo calves can be successfully weaned as early as 8 weeks of age without negatively affecting their growth performance.

Physical activity and associated factors among young adults in Malaysia: an online exploratory survey.

The burden of non-communicable diseases is increasing in Malaysia. Insufficient Physical Activity, which is an important risk factor for non-communicable diseases, is less researched in Malaysia. We aimed to assess the level of physical activity and identify its correlates. An online survey was carried out during October, 2011 in the University Tunku Abdul Rahman by the opinion poll research committee. Young adults answered the Short International Physical Activity Questionnaire and a questionnaire about factors according to a socio-ecological model which was adapted from published studies. Metabolic equivalent (MET)-hours and MET-minutes were calculated. Physical activity was classified as sufficient when MET-minutes were > 840. The mean age of the 474 participants was 22.4 years (S.D. = 4.7), and 253 (53.4%) were females. Their mean and median of MET-hours of PA done during the previous seven days were 31.36 (S.D., 52.19) and 14.7 (IQR, 5.77-32.07), respectively. Physical activity done was sufficient among 242 (51.1%) participants. Using univariate analysis, being male, good self-rated health, positive intention, self-efficacy, perceived benefits, social support, and availability of facilities were associated with sufficient physical activity. Using multivariate analysis sufficient physical activity was associated with participants' intention (OR 0.75, 95% CIs 0.64, 0.88), self-efficacy (OR 0.91, 95% CIs 0.85, 0.97) and facility availability (OR 0.81, 95% CIs 0.73, 0.91). The proportion of participants with sufficient physical activity was low. Positive intention and self-efficacy associated with sufficient physical activity should be supported by availability of facilities and a safely-built environment. A nationwide survey about physical and associated socialecological factors is needed to design rational health promotion strategies.

A note on water quality associated with slaughter premises in Bangladesh.

Water quality used for washing carcasses and abattoir premises was assessed at two sites in southeast Bangladesh. In addition, the effect of discharging untreated washings from slaughter premises on water purity in the receiving waterways was assessed from pH, dissolved oxygen and electrical conductivity measurements. Premises effluents did not have a significant effect on the physico-chemical properties of the water in the receiving canals, as determined from the difference between the 4 m upstream and 4 m downstream samples, relative to the entry point of the effluent. When a water shortage occurred at one of the premises, water was sourced for washing the carcasses and premises from a canal that was also used for conveying municipal effluent. The discharge water from the premises at this time had a pH, dissolved oxygen content and conductivity of 7.93, 1.84 mg/L and 6.06 mS/cm respectively, and this would be a threat to survival of freshwater animal life.

Sensory evaluation and consumer acceptance of eggs from hens fed flax seed and 2 different antioxidants.

The sensory attributes and consumer acceptance of eggs from flax seed-fed hens were evaluated by trained and untrained panelists. Hens were fed diets containing 0% flax seed (control), 10% flax seed (flax), 10% flax seed+100 IU/kg of vitamin E (flax+α-tocopherol), or 10% flax seed+100 mg/kg of butylated hydroxytoluene (BHT) (flax+BHT). Fresh eggs collected within 24 h were hard-boiled, coded, and were offered to trained panelists in 4 testing sessions. Sensory traits evaluated were aroma, flavor, off-flavor, and overall difference. The trained panelists rated flax, flax+α-tocopherol, and flax+BHT eggs to be different from control eggs (P<0.001). In a second study, sensory attributes were tested by untrained panelists. The majority (75 to 80%) of the panelists could not distinguish flax seed-fed versus control eggs for aroma and flavor. A consumer preference test was also conducted to gauge end-user response to flax seed-fed eggs. Consumer acceptance testing did not find any significant difference (P>0.05) between control and flax seed-fed eggs. These results suggest that flax seed when incorporated at 10% in the layer diet can produce eggs that are acceptable to untrained panelists and consumers. However, trained panelists are able to detect differences in flavor, aroma, and off-flavor and overall difference in eggs from hens fed flax seed. Antioxidant supplementation (vitamin E, BHT) did not enhance the acceptability of flax seed-fed eggs by trained panelists.

Skin injuries identified in cattle and water buffaloes at livestock markets in Bangladesh.

Skin injuries were assessed in 560 imported and local cattle and water buffaloes at two livestock markets in Bangladesh. The body of each animal was divided into 11 anatomical regions, and abrasions, lacerations, penetrations, ulcerations, bleeding, swelling, hyperkeratosis and scars were recorded for each region. Among the 560 animals studied, 501 were found to have at least one injury. The prevalence of skin injuries was 89 per cent, with 84 per cent of the cattle and 99 per cent of the water buffaloes having obvious skin injuries. The most common types of injury were abrasions that were found in 73 per cent of the animals, followed by scars (50 per cent), and lacerations (41 per cent). Buffaloes had more abrasions (95 per cent), lacerations (57 per cent), swelling (15 per cent) and hyperkeratosis (32 per cent) compared with cattle, whereas scars (60 per cent) were more common in cattle (P<0.001). Within the 11 different anatomical regions, all types of injuries were present but in different proportions. The buttock region had a higher proportion of abrasions (36 per cent) followed by the hip, hindlimb and back regions. Penetration, ulceration, bleeding and swelling were present at lower frequencies in all regions. Causes for these injuries included rubbing against the inside wall of vehicles used for transportation and stock-handler abuse (59 per cent and 13 per cent, respectively). Buffaloes sustained more transport injuries than cattle, and the number of injuries was higher in imported than local animals.

Oxidative stability and lipid components of eggs from flax-fed hens: effect of dietary antioxidants and storage.

An experiment was conducted to investigate the effect of dietary antioxidants and storage on fatty acid profile, oxidative stability, and vitamin E concentration of n-3 fatty acid-enriched eggs. Eggs (384, 48/diet) were collected from ISA Brown layers fed diets containing corn-soy (control) with 100 g/kg of flax seed and 2 types of antioxidants [alpha-tocopherols (alpha-TOC) and butylated hydroxytoluene (BHT)] at 0, 50, 100, or 150 IU or mg/kg. Eggs were stored at 4 degrees C. On d 0, 20, 40, and 60 of storage, 2 eggs were selected randomly from each replicate (totaling 12 eggs per treatment) and analyzed. Eggs from hens fed flax had increased alpha-linolenic (18:3n-3), eicosapentaenoic (20:5n-3), and docosahexaenoic acids (DHA, 22:6n-3) and decreased arachidonic acid (20:4n-6) and total n-6:n-3 ratio when compared with control eggs (P<0.05). The n-6:n-3 fatty acid ratio was lowest in the flax+50 IU of alpha-TOC, flax+100 IU of alpha-TOC, and flax+BHT supplemented group when compared with the flax group (P<0.05). With the exception of flax+100 mg of BHT, addition of antioxidants led to a reduction in palmitic acid in fresh eggs (P<0.05). During the first 20 d of storage, over a 17% reduction in total n-3 fatty acids was observed in eggs from flax+50 mg of BHT supplemented groups (P<0.05). Docosahexaenoic acid was the predominant long-chain n-3 fatty acid in egg and was stable during storage in the control, flax, flax+100 IU of alpha-TOC, flax+150 IU of alpha-TOC, and flax+150 mg of BHT groups. However, antioxidant supplementation had no effect on DHA upon storage in flax+50 IU of alpha-TOC and flax+50 mg of BHT eggs where over 13 to 17% reduction in DHA content was observed during 20 to 60 d of storage (P<0.05). Inclusion of alpha-TOC led to over 4.5- to 12-fold increases in alpha-TOC in eggs. Egg storage for 40 d or longer led to over 50% reduction in egg alpha-TOC (P<0.05). Feeding flax seeds led to an increase in TBA reactive substances in eggs (P<0.05). alpha-Tocopherol was better in preventing lipid oxidation than BHT at d 0 of storage. However, neither had a significant effect on egg TBA reactive substances upon 60 d of storage (P>0.05). These studies demonstrate that the level and type of antioxidants and duration of egg storage significantly affected the fatty acid profile, alpha-TOC status, and oxidative stability of chicken eggs.

Association of lipid profile with ischemic stroke.

This study is an observational case control study conducted in the Neurology department of Dhaka Medical college Hospital (DMCH) to see the relation of ischemic stroke with different components of serum lipids. Cases and controls were selected following certain inclusion and exclusion criteria. Result showed that ischemic stroke was more common after the age of 50 years with mean age of 63.58+/-10.22 years. Male suffered more than female from ischemic stroke (M:F=2.57:1). 80% of the stroke patients belong to middle class economic condition who avail free services of Govt. hospitals. Hypertension and diabetes mellitus (DM) were found to be significant risk factors for ischemic stroke (P<0.01 and P<0.05 respectively). High level of serum total cholesterol and low density lipoprotein (LDL) cholesterol showed significant risk in ischemic stroke (p<0.05). In contrast, low level of high density lipoprotein (HDL) cholesterol appeared as a significant risk factor (p<0.01) indicating beneficial effect of HDL cholesterol on atherosclerotic process. Serum triglyceride level showed no significant effect on ischemic stroke (p>0.05).

Improvement of liquid and frozen-thawed semen quality of Nili-Ravi buffalo bulls (Bubalus bubalis) through supplementation of fat.

The aim of the study was to investigate the effects of dietary fat on quality of liquid and frozen-thawed semen of Nili-Ravi buffalo bulls. Adult bulls (n=21) were fed a balanced ration (Con; n=7) or the same ration either containing sunflower oil (SF-O; n=7) or whole sunflower seeds (SF-S; n=7) for 63 days. Body weight and body condition score of each bull was recorded on days 0, 30 and 60 of the experiment. Semen was collected on days 39, 46, 53 and 60, frozen by a fast method and stored at -196 degrees C for 24h. Sperm motility was assessed using a bright field microscope. Plasma membrane integrity of fresh and frozen-thawed spermatozoa was assessed using a hypo-osmotic swelling (HOS) assay. The concentration of spermatozoa and volume of semen was not different among groups on various days of collection. Sunflower-enriched diets did not affect the motility and number of HOS-positive spermatozoa in the fresh semen. Motility and HOS of post-thawed spermatozoa were higher (p<0.05) in bulls fed the sunflower-enriched diets. Similarly, diets did not affect the body condition score and body weight of bulls. In conclusion, feeding of sunflower oil or sunflower seed as fat sources can improve the quality of buffalo bull spermatozoa.

Kinetics of batch single cell protein production from rice polishings with Candida utilis in continuously aerated tank reactors.

Single cell protein was produced from the defatted rice polishings by fermentation with Candida utilis in an aerated 14-L fermentor to optimize bioprocess variables. Maximum values of specific growth rate coefficient (mu, h(-1)), cell mass yield (Y(X/S), g/g) and cell mass productivity (g/Lh) were 0.31, 0.65, and 1.24, respectively under optimized conditions of aeration rate (1 v.v(-1) m(-1)), dissolved oxygen (50%), corn steep liquor (5%), temperature (35 degrees C), and substrate concentration (90 g rice polishings/L) in yeast salt medium (pH 6.0). The kinetic parameters for 50-L fermentor under same conditions were 0.33 h(-1), 0.66 g/g, 1.33 g/Lh, 2.25 g/Lh, 1.23 g/Lh, 0.45 g/g substrate and 0.20 g/g cell h for mu, Y(X/S), Q(X), Q(S), Q(CP), Y(TP/S), and q(CP), respectively and were significantly higher than their respective values reported on C. utilis in batch culture studies. This biomass protein contained 23.6%, 32.75%, 11.50%, 12.95%, 10.5%, and 0.275% true protein, crude protein, crude fiber, ash, cellulose and RNA content respectively. This implied that the fermentation process could be up scaled to manufacture animal feed. Gross metabolizable energy content of dried SCP was 29,711 kcal/kg and indicated that the SCP could serve both as energy as well as a protein source. Yeast can replace expensive feed ingredients currently being incorporated in poultry feed and can reduce cost of poultry ration by 0.33 US dollars-0.51 US dollars/100 kg bag and improve the economics of feed production in our country.

Dihydropyrimidine dehydrogenase deficiency presenting at birth.

Dihydropyrimidine dehydrogenase (DPD) deficiency (McKusick 274270) is a clinically heterogeneous autosomal recessive disorder of pyrimidine metabolism. DPD is the enzyme that catalyses the first and the rate-limiting step in the catabolism of uracil, thymine and the analogue 5-fluorouracil. To date, more than 30 patients have been diagnosed with a complete enzyme deficiency. Here, we describe the fifth case with a complete DPD deficiency presenting at birth with severe neurological abnormalities. The patient was homozygous for the common splice-site mutation IVS14+1G > A.

Influence of minerals on the aetiology of geophagia in periurban dairy cattle in the derived savannah of Nigeria.

The aetiology of geophagia in periurban dairy cattle in Nigeria was assessed in relation to the mineral status of the serum, the herbage consumed, and the soil from where the herbage was consumed. The study was carried out using nine herds in the derived savannah zone during both the wet and dry seasons. Generally, the Na, Ca, Fe, Zn and Cu levels in the samples were adequate. However, the forages were low in Ca and the serum was deficient in P. Forage, soil and termite hill samples were also deficient in P. The low levels of P in the soil and termite hill samples showed that the consumption of sand by the cattle did not have any positive impact on their P intake. A seasonal effect was apparent (p < 0.05) on the serum Na+, PO4(3-), Cu2+ and Fe2+ concentrations but not on Ca2+ and Zn2+ concentrations. A seasonal effect was also significant (p < 0.05) on the minerals in the forage. There were also seasonal differences in most of the soil and termite hill minerals. Most samples had higher (p < 0.05) mineral levels in the dry season. Herd size and farming activity had no apparent influence on the aetiology of geophagia as they did not affect (p > 0.05) the concentration of P in the serum, the reported cause of geophagia. Geophagia is probably caused by a marginal level of P in the serum and low levels in the forage and soil. Experimental trials will be required to confirm these observations.

Insulin therapy for a non-diabetic patient with severe hypertriglyceridemia.

OBJECTIVE: To compare the short and long term effectiveness of fish oil, insulin, and gemfibrozil in a non-diabetic patient with severe hypertriglyceridemia. METHOD: An adolescent male with hypertriglyceridemia (triglyceride level 4575 mg/dl) and abdominal pain was treated with the goal of immediate reduction and maintenance of triglyceride (TG) level below 1000 mg/dl. Fish oil, insulin and gemfibrozil were administered sequentially, in separate time blocks, for a duration of 3, 6, and 6 months, respectively. RESULTS: Fish oil took several weeks to lower TG level, and patient compliance during 3 months of therapy was inadequate. Insulin was effective in immediately lowering the TG level, but was unable to maintain the level below 1000 mg/dl. Gemfibrozil was ineffective in achieving the immediate reduction of TG level; however, it was adequate in maintaining the desired level in the long-term and patient compliance was better than with the fish oil. CONCLUSION: In patients with risk of pancreatitis due to severe hypertriglyceridemia, immediate reduction of the triglyceride level is achievable by using a single dose of regular insulin (0.1 unit/kg, subcutaneous) while long-term maintenance therapy can be provided by gemfibrozil.

Mutational analysis of the human immunodeficiency virus type 1 Vpu transmembrane domain that promotes the enhanced release of virus-like particles from the plasma membrane of mammalian cells.

Human immunodeficiency virus type 1 Vpu is a multifunctional phosphoprotein composed of the N-terminal transmembrane (VpuTM) and C-terminal cytoplasmic domains. Each of these domains regulates a distinct function of the protein; the transmembrane domain is critical in virus release, and phosphorylation of the cytoplasmic domain is necessary for CD4 proteolysis. We carried our experiments to identify amino acids in the VpuTM domain that are important in the process of virus-like particle (VLP) release from HeLa cells. VLPs are released from the plasma membrane of HeLa cells at constitutive levels, and Vpu expression enhanced the release of VLPs by a factor of 10 to 15. Deletion of two to five amino acids from both N- and C-terminal ends or the middle of the VpuTM domain generated mutant Vpu proteins that have lost the ability to enhance VLP release. These deletion mutants have not lost the ability to associate with the wild-type or mutant Vpu proteins and formed complexes with equal efficiency. They were also transported normally to the Golgi complex. Furthermore, a Vpu protein having the CD4 transmembrane and Vpu cytoplasmic domains was completely inactive, and Vpu proteins harboring hybrid Vpu-CD4 TM domains were also defective in the ability to enhance the release of VLPs. When tested for functional complementation in cotransfected cells, two inactive proteins were not able to reconstitute Vpu activity that enhances the release of Gag particles. Coexpression of functional CD4/Vpu hybrids or wild-type Vpu with inactive mutant CD4/Vpu proteins revealed that mutations in the VpuTM domain could dominantly interfere with Vpu activity in Gag release. Taken together, these results demonstrated that the structural integrity of the VpuTM domain is critical for Vpu activity in the release of VLPs from the plasma membrane of mammalian cells.

Abnormal thyroid function tests in infants with congenital hypothyroidism: the influence of soy-based formula.

OBJECTIVE: To assess the etiology of hyperthyroxinemia or hyperthyrotropinemia in infants with congenital hypothyroidism who are on replacement therapy with L-thyroxine. METHODS: These infants were treated with recommended doses of L-thyroxine following the diagnosis of congenital hypothyroidism. Because of hyperthyroxinemia (2 patients) and hyperthyrotropinemia (1 patient), medication compliance and dietary practice (formula type, age of introduction, and discontinuation or change of the formula) were assessed. Clinical evaluation was also performed. RESULTS: Elevated thyroxine level in 2 infants was associated with discontinuation of soy formula 4 weeks previously; reduction of L-thyroxine dose normalized serum levels in both of these infants. In the third infant, who received soy formula from 1 week of age, TSH remained elevated despite incremental L-thyroxine doses of 19 micrograms/kg/day; discontinuation of soy formula was followed by normalization of the TSH in 3 weeks and helped attain a subsequent decrement of L-thyroxine dose to 8.6 micrograms/kg/day. Neither the hyperthyroxinemia nor hyperthyrotropinemia in these infants was associated with any adverse behavioral-developmental consequence. CONCLUSION: When initiating soy-formula feeding in infants with congenital hypothyroidism, the L-thyroxine dose should be increased because of significant reduction in intestinal absorption: conversely, when soy feeding is discontinued, the L-thyroxine dose should be decreased.

Phosphorylation of both phosphoacceptor sites in the HIV-1 Vpu cytoplasmic domain is essential for Vpu-mediated ER degradation of CD4.

Human immunodeficiency virus type 1 (HIV-1) Vpu is phosphorylated at two serine residues (Ser52 and Ser56) present within the acidic dodecapeptide region of the 54-aa cytoplasmic domain. Previous experiments have shown that Vpu phosphorylation is critical for the degradation of CD4 in the endoplasmic reticulum. In this study, we carried out experiments to elucidate the role of individual phosphoacceptor sites in CD4 proteolysis. We show here that acidic amino acids could not functionally substitute for phosphoserines in Vpu that is capable of inducing the degradation of CD4. Our studies have further revealed that phosphorylation of either of the two phosphoacceptor sites is not sufficient to generate a functional Vpu protein. When tested for functional complementation, inactive phosphorylation-proficient Vpu mutants failed to generate Vpu proteins that had the ability to induce the degradation of Vpu-sensitive glycoproteins. The failure to complement was not due to assembly defects in the Vpu protein as unphosphorylated Vpu formed oligomeric complexes in the cell. We also showed that Vpu expression inhibits protein transport in a phosphorylation-dependent manner. Our studies have thus revealed that both phosphoserines in Vpu are critical participants in a pathway that leads to the proteolysis of CD4 in the ER and that these phosphoserines should be present on the same subunit of the Vpu protein.