SARS-CoV-2 mitochondrial metabolic and epigenomic reprogramming in COVID-19.

To determine the effects of SARS-CoV-2 infection on cellular metabolism, we conducted an exhaustive survey of the cellular metabolic pathways modulated by SARS-CoV-2 infection and confirmed their importance for SARS-CoV-2 propagation by cataloging the effects of specific pathway inhibitors. This revealed that SARS-CoV-2 strongly inhibits mitochondrial oxidative phosphorylation (OXPHOS) resulting in increased mitochondrial reactive oxygen species (mROS) production. The elevated mROS stabilizes HIF-1α which redirects carbon molecules from mitochondrial oxidation through glycolysis and the pentose phosphate pathway (PPP) to provide substrates for viral biogenesis. mROS also induces the release of mitochondrial DNA (mtDNA) which activates innate immunity. The restructuring of cellular energy metabolism is mediated in part by SARS-CoV-2 Orf8 and Orf10 whose expression restructures nuclear DNA (nDNA) and mtDNA OXPHOS gene expression. These viral proteins likely alter the epigenome, either by directly altering histone modifications or by modulating mitochondrial metabolite substrates of epigenome modification enzymes, potentially silencing OXPHOS gene expression and contributing to long-COVID.

The anti-HIV drug abacavir stimulates ß-catenin activity in osteoblast lineage cells.

Bone mineral density (BMD) loss in people living with HIV occurs with the initiation of combined antiretroviral therapy (cART), particularly with tenofovir disoproxil fumarate (TDF) containing cART. Switching from TDF to abacavir (ABC) or dolutegravir (DTG) leads to increased BMD. Whether BMD gains are due to cessation of TDF or anabolic effects of ABC or DTG is unclear. We investigated the effects of ABC and DTG on osteoblast lineage cells in vitro and in vivo. Primary human osteoblasts and male C57BL/6 mice were treated with individual antiretrovirals (ARVs) or a combination of ABC/DTG/lamivudine (3TC). Nearly all ARVs and cART inhibited osteogenic activity in vitro. Due to the importance of Wnt/ß-catenin in bone formation, we further investigated ARV effects on the Wnt/ß-catenin pathway. ABC, alone and as part of ABC/DTG/3TC, increased osteoblastic ß-catenin activity as indicated by increased TOPFlash activity, hypo-phosphorylated (active) ß-catenin staining, and ß-catenin targeted gene expression. Mice treated with TDF had decreased lumbar spine BMD and trabecular connectivity density in the vertebrae, while those treated with ABC/DTG/3TC reduced cortical area and thickness in the femur. Mice treated with ABC alone had no bone structural changes, increased circulating levels of the bone formation marker, P1NP, and elevated expression of the Wnt/ß-catenin target gene, Lef1, in osteocyte enriched samples. Further, bones from ARV-treated mice were isolated to evaluate ARV distribution. All ARVs were detected in the bone tissue, which was inclusive of bone marrow, but when bone marrow was removed, only TDF, ABC, and DTG were detected at ~0.1% of the circulating levels. Overall, our findings demonstrate that ABC activates Wnt/ß-catenin signaling, but whether this leads to increased bone formation requires further study. Assessing the impact of ARVs on bone is critical to informing ARV selection and/or discovery of regimens that do not negatively impact the skeleton.

An Efficient Humanized Mouse Model for Oral Anti-Retroviral Administration.

HIV anti-retrovirals (ARVs) have vastly improved the life expectancy of people living with HIV (PLWH). However, toxic effects attributed to long-term ARV use also contribute to HIV-related co-morbidities such as heart disease, bone loss and HIV-associated neurocognitive disorders (HAND). Unfortunately, mouse models used to study the effects of ARVs on viral suppression, toxicity and HIV latency/tissue reservoirs have not been widely established. Here, we demonstrate an effective mouse model utilizing immune-compromised mice, reconstituted with infected human peripheral blood mononuclear cell (PBMCs). ARVs areincorporated into mouse chow and administered daily with combination ARV regimens includingAtripla (efavirenz, tenofovir disoproxil fumarate, and emtricitabine) and Triumeq (abacavir, dolutegravir and lamivudine). This model measures HIV-infected human cell trafficking, and ARV penetration throughout most relevant HIV organs and plasma, with a large amount of trafficking to the secondary lymphoid organs. Furthermore, the HIV viral load within each organ and the plasma was reduced in ARV treated vs. untreated control. Overall, we have demonstrated a mouse model that is relatively easy and affordable to establish and utilize to study ARVs' effect on various tissues, including the co-morbid conditions associated with PLWH, such as HAND, and other toxic effects.

Brief Report: Undercarboxylated Osteocalcin Is Associated With Cognition in Women With and Without HIV.

INTRODUCTION: Bone loss and cognitive impairment are common in women living with HIV (WLWH) and are exacerbated by menopause. Bone-derived undercarboxylated osteocalcin (ucOCN) and sclerostin appear to influence cognition. The current study investigated whether the circulating levels of these 2 proteins are associated with cognition in midlife WLWH and demographically similar HIV seronegative women. METHODS: Plasma samples from women enrolled in a musculoskeletal substudy within the Women's Interagency HIV Study were used to measure ucOCN and sclerostin. A neuropsychological (NP) test battery assessing executive function, processing speed, attention/working memory, learning, memory, verbal fluency, and motor function was administered within 6 months of musculoskeletal enrollment and every 2 years after (1-4 follow-up visits per participant). A series of generalized estimating equations were conducted to examine the association between biomarkers and NP performance at the initial assessment and over time in the total sample and in WLWH only. Primary predictors included biomarkers, time, and biomarker by time interactions. If the interaction terms were not significant, models were re-run without interactions. RESULTS: Neither biomarker predicted changes in NP performance over time in the total sample or in WLWH. ucOCN was positively associated with executive function in the total sample and in WLWH and with motor skills in WLWH. ucOCN was negatively associated with attention/working memory in the total sample. There were no significant associations between sclerostin and NP performance. CONCLUSION: The current study suggests an association between bone-derived ucOCN and cognition in women with and without HIV infection.

Bone Quality in Relation to HIV and Antiretroviral Drugs.

PURPOSE OF REVIEW: People living with HIV (PLWH) are at an increased risk for osteoporosis, a disease defined by the loss of bone mineral density (BMD) and deterioration of bone quality, both of which independently contribute to an increased risk of skeletal fractures. While there is an emerging body of literature focusing on the factors that contribute to BMD loss in PLWH, the contribution of these factors to bone quality changes are less understood. The current review summarizes and critically reviews the data describing the effects of HIV, HIV disease-related factors, and antiretroviral drugs (ARVs) on bone quality. RECENT FINDINGS: The increased availability of high-resolution peripheral quantitative computed tomography has confirmed that both HIV infection and ARVs negatively affect bone architecture. There is considerably less data on their effects on bone remodeling or the composition of bone matrix. Whether changes in bone quality independently predict fracture risk, as seen in HIV-uninfected populations, is largely unknown. The available data suggests that bone quality deterioration occurs in PLWH. Future studies are needed to define which factors, viral or ARVs, contribute to loss of bone quality and which bone quality factors are most associated with increased fracture risk.

Anti-HIV Drugs Cause Mitochondrial Dysfunction in Monocyte-Derived Macrophages.

Combination antiretroviral therapy (cART) dramatically changed the face of the HIV/AIDS pandemic, making it one of the most prominent medical breakthroughs of the past 3 decades. However, as the life span of persons living with HIV (PLWH) continues to approach that of the general population, the same cannot be said regarding their quality of life. PLWH are affected by comorbid conditions such as high blood pressure, diabetes, and neurocognitive impairment at a higher rate and increased severity than their age-matched counterparts. PLWH also have higher levels of inflammation, the drivers of which are not entirely clear. As cART treatment is lifelong, we assessed here the effects of cART, independent of HIV, on primary human monocyte-derived macrophages (MDMs). MDMs were unskewed or skewed to an alternative phenotype and treated with Atripla or Triumeq, two first-line cART treatments. We report that Triumeq skewed alternative MDMs toward an inflammatory nonsenescent phenotype. Both Atripla and Triumeq caused mitochondrial dysfunction, specifically efavirenz and abacavir. Additionally, transcriptome sequencing (RNA-seq) demonstrated that both Atripla and Triumeq caused differential regulation of genes involved in immune regulation and cell cycle and DNA repair. Collectively, our data demonstrate that cART, independent of HIV, alters the MDM phenotype. This suggests that cART may contribute to cell dysregulation in PLWH that subsequently results in increased susceptibility to comorbidities.

Bone and fat hormonal crosstalk with antiretroviral initiation.

BACKGROUND: Bone mineral density (BMD) loss and fat gain is common in people living with HIV (PLWH), particularly after initiating combination antiretroviral therapy (cART). Given the close metabolic interaction between bone and fat, we tested the hypotheses that changes in bone-derived hormones are associated with fat accumulation and changes in fat-derived hormones are associated with BMD loss following cART initiation. METHODS: HIV-seropositive subjects (n = 15) initiating fixed dose cART of tenofovir disoproxil fumarate/emtricitabine/efavirenz (TDF/FTC/EFV) underwent dual X-ray absorptiometry (DXA) assessment pre-cART and again 12-months post-cART initiation. DXA-derived measurements included BMD at the lumbar spine, femoral neck, total hip, and trochanter and the trunk and total fat. Serum undercarboxylated osteocalcin (ucOCN), sclerostin, lipocalin-2, leptin, and adiponectin were measured pre and post-cART. Spearman's rank-order correlations assessed the cross-sectional associations between hormones and bone and fat mass pre- and post-cART. Linear regression models adjusting for baseline bone or fat mass assessed the association between hormone change and BMD/fat changes following cART initiation. RESULTS: ucOCN (p = 0.04) and lipocalin-2 (p = 0.03) increased post-cART while sclerostin, leptin, and adiponectin remained unchanged. BMD significantly decreased post-cART at all skeletal sites. Trunk and total fat increased post-cART but not significantly, while weight and BMI remained unchanged. In models adjusting for baseline BMD and fat mass, change in ucOCN was negatively associated with change in trunk (p = 0.008) and total fat (p = 0.01) and the change in leptin was positively associated with change in total hip (p = 0.03) and trochanteric BMD (p = 0.02). CONCLUSION: The current study demonstrates bone-fat crosstalk in cART initiating PLWH.

Change in Circulating Undercarboxylated Osteocalcin (ucOCN) Is Associated With Fat Accumulation in HIV-Seropositive Women.

BACKGROUND: Bone mineral density loss and fat accumulation are common in people living with HIV. The bone-derived hormone, undercarboxylated osteocalcin (ucOCN) regulates fat metabolism. We investigated the relationship between ucOCN change and body fat change among perimenopausal/postmenopausal HIV-seronegative and HIV-seropositive women on long-term antiretrovirals. METHODS: Perimenopausal and postmenopausal women enrolled in the Women's Interagency HIV Study MSK substudy underwent trunk and total fat assessment by dual energy x-ray absorptiometry (DXA) at study enrollment (index visit) and again 2 years later. Circulating ucOCN and cOCN were also measured at the index and 2-year visits. The correlation between the 2-year change in ucOCN and cOCN and change in trunk and total fat was assessed as a function of HIV serostatus using linear regression modeling. Multivariate linear regression assessed the association between ucOCN and cOCN change and total and trunk fat change after adjusting for sociodemographic variables. Linear regression models restricted to HIV-seropositive women were performed to examine the contributions of HIV-specific factors (index CD4 count, viral load, and combined antiretroviral therapy use) on the associations. RESULTS: Increased ucOCN over the 2-year follow-up was associated with less trunk and total fat accumulation in models adjusting for HIV serostatus and participants sociodemographics, whereas there was no association with cOCN and the fat parameters. None of the HIV-specific factors evaluated influenced the association between ucOCN and fat parameters. CONCLUSION: The current study suggests that increases in ucOCN are associated with decreased fat accumulation in HIV-seronegative and HIV-seropositive postmenopausal women on long-term antiretroviral therapy.

Extended stability range of the non-Fermi liquid phase in UCoAl.

High pressure was used to investigate the stability of the non-Fermi liquid (NFL) state, observed in electrical resistivity of uranium-based band metamagnet UCoAl in a pure form (paramagnet) or with Fe substitution (ferromagnetic ground state), both in a single-crystal form. By combining the pressure variations of magnetization and resitivity in these materials the phase diagram for UCoAl had been constructed. The band metamagnet transforms into the ferromagnetic state as the critical metamagnetic field is reduced to zero by the lattice expansion analogous to the negative pressure. Within the same diagram, the increasing hydrostatic pressure drives the critical metamagnetic field upwards while reducing the magnetization increment at the transition. The NFL state persists to about 4-5 GPa. Although spin fluctuations play an important role in the character of UCoAl, they do not exhibit any criticality in the sense of divergence of parameters describing the resistivity around the Ferro-NFL phase transition, which is of the first order type.

A Clinical Trial on Weight Loss among Truck Drivers.

BACKGROUND: The high prevalence of obesity among commercial truck drivers may be related to sedentary nature of the job, lack of healthy eating choices, and lack of exercise. There may be a link between obesity and crash risk, therefore an intervention to reduce obesity in this population is needed. OBJECTIVE: To assess feasibility of a 12-week weight loss intervention for truck drivers with a weight loss goal of 10% of initial body weight. METHODS: Drivers were selected based on age (≥21 years) and body mass index (≥30 kg/m^2). The drivers participated in a before-after clinical trial. The intervention included a 12-week program that provided information on healthy diet and increasing exercise, and telephone-based coaching using SMART goals. Outcomes included change from baseline in reported energy intake, measured weight, waist, hip, and neck circumference, blood pressure, and point of care capillary blood lipids and hemoglobin A1c. Exit interviews were conducted to gain insight into driver opinions on the program features and usefulness. This study was registered with the NIH Clinical Trials Registry, number NCT02348983. RESULTS: 12 of 13 drivers completed the study. Weight loss was statistically significant (p=0.03). Reported energy (p=0.005), total fat consumption (p=0.04), and saturated fat consumption (p=0.02) intake were also lower after the 12-week intervention. Drivers attributed their weight loss to health coaching and suggested a longer intervention so that they could reach their goal and become accustomed to the changes. CONCLUSION: This weight loss intervention is feasible for this difficult population. Additional research is needed to compare this intervention with a control group.

High-field magnetization and magnetoelasticity of single crystalline HoFe5Al7.

Magnetization and ultrasound measurements have been performed in pulsed magnetic fields up to 60 T on a ferrimagnetic HoFe5Al7 single crystal (Curie temperature TC = 216 K, compensation point Tcomp = 65 K) with a tetragonal crystal structure of the ThMn12-type. The compound exhibits a high magnetic anisotropy of the easy-plane type. A large anisotropy is also observed within the basal plane having an easy-magnetization direction along the [110] axis with the spontaneous magnetic moment Ms = 2 µB/f.u. at T = 2 K. Along the easy axis, two first-order field-induced magnetic transitions are observed. At both transitions sharp anomalies in the acoustic properties are found. The critical fields of the transitions depend on temperature in a different manner. Within molecular-field theory and using the high-field magnetization data the Ho-Fe inter-sublattice exchange parameter has been determined to be nHoFe ≈ 4 T/µB. The magnetoelasticity has also been probed by magnetization measurements under hydrostatic pressure. TC decreases with a rate dTC/dp = -10 K/GPa, whereas Tcomp increases with dTcomp/dp = 3.5 K/GPa.

Pressure dependence of magnetic properties in Fe-Mn-B amorphous alloys: evidence for inhomogeneous ferromagnetism.

The pressure dependence of the saturation magnetization and Curie temperature was studied in melt-spun Fe60Mn20B20, Fe56Mn24B20 and Fe75B25 amorphous alloys up to 0.9 GPa, corresponding to volume changes up to 0.45%. In addition, in situ high-pressure (up to 40 GPa) x-ray diffraction was performed to determine the compressibility of the latter two alloys. Both the Curie temperature TC (at atmospheric pressure TC = 201 ± 3 and 159 ± 3 K) and the low-temperature saturation magnetization M5 K,5 T decrease remarkably with increasing pressure: dTC/dp =- 31 ± 0.5 and -32 ± 5 K GPa(-1) and dlnM5 K,5 T/dp =- 0.15 ± 0.02 and -0.13 ± 0.03 GPa(-1) for xMn = 20 and 24 at.%, respectively. Compared to dlnM5 K,5 T/dp =- 0.016 ± 0.003 GPa(-1) measured for Fe75B25, the pressure dependence of M5 K,5 T is one order of magnitude larger in the ternary alloys. The bulk moduli for the Fe56Mn24B20 and Fe75B25 glasses were measured to be 152 GPa and 173 GPa, respectively. These data are also compared with the pressure dependence of the hyperfine field and theoretical calculations of the saturation moment for Fe-B alloys reported in the literature. The results were interpreted within an inhomogeneous itinerant-electron model of ferromagnetism.

Practical system for the direct measurement of magneto-caloric effect by micro-thermocouples.

A system for direct measurements of the magneto-caloric effect (MCE) exploits a rapid transport of a sample into or from magnetic field in permanent Halbach-type (1 T) or superconducting (4.7 T) magnets. Time dependence of induced changes of the sample temperature, ΔT(t), is detected directly by the differential Cu-Constantan-Cu micro-thermocouples with time steps of 300 ms. A sample placed inside an evacuated simple LN(2) cryostat is either totally isolated (adiabatic conditions) or partly connected with the copper sample holder (non-adiabatic conditions). The last arrangement (a model of the Brayton cycle) is used to simulate an application of MCE in refrigeration techniques. The relations describing ΔT(t) that allow an analysis of MCE of the studied materials are based on the general cooling law. The effect of the first-order magnetic transition on MCE of selected sample is also demonstrated by non-standard ΔT(t) curves measured in the last mentioned experimental arrangements.

Exchange bias in bulk layered hydroxylammonium fluorocobaltate (NH3OH)2CoF4.

The magnetic properties of layered hydroxylammonium fluorocobaltate (NH(3)OH)(2)CoF(4) were investigated by measuring its dc magnetic susceptibility in zero-field-cooled (ZFC) and field-cooled (FC) regimes, its frequency dependent ac susceptibility, its isothermal magnetization curves after ZFC and FC regimes, and its heat capacity. Effects of pressure and magnetic field on magnetic phase transitions were studied by susceptibility and heat capacity measurements, respectively. The system undergoes a magnetic phase transition from a paramagnetic state to a canted antiferromagnetic state exhibiting a weak ferromagnetic behavior at T(C) = 46.5 K and an antiferromagnetic transition at T(N) = 2.9 K. The most spectacular manifestation of the complex magnetic behavior in this system is a shift of the isothermal magnetization hysteresis loop in a temperature range below 20 K after the FC regime-an exchange bias phenomenon. We investigated the exchange bias as a function of the magnetic field during cooling and as a function of temperature. The observed exchange bias was attributed to the large exchange anisotropy which exists due to the quasi-2D structure of the layered (NH(3)OH)(2)CoF(4) material.

Effect of pressure on the magnetic properties of TM(3)[Cr(CN)(6)](2)·12H(2)O.

We present the results of magnetization and AC susceptibility measurements performed on ferrimagnetic Mn(3)(2+)[Cr(III)(CN)(6)](2)·12H(2)O and ferromagnetic Ni(3)(2+)[Cr(III)(CN)(6)](2)·12H(2)O systems under pressures up to 0.9 GPa in a commercial SQUID magnetometer. The magnetization process is affected by pressure: magnetization saturates at higher magnetic field, saturated magnetization µ(s) of Ni(3)[Cr(CN)(6)](2) is reduced and almost unaffected for Mn(3)[Cr(CN)(6)](2) at low temperatures. The Curie temperature T(C) of Mn(3)[Cr(CN)(6)](2) increases with the applied pressure, ΔT(C)/Δp = 25.5 K GPa(-1), due to a strengthened super-exchange antiferromagnetic interaction J(AF), but it is not affected significantly in the case of Ni(3)[Cr(CN)(6)](2) with a dominant ferromagnetic J(F) super-exchange interaction. The increase in the J(AF) interaction is attributed to the enhanced value of the single electron overlapping integral S and the energy gap Δ of the mixed molecular orbitals t(2g) (Mn(2+)) and t(2g) (Cr(III)) induced by pressure.

Helimagnetism of Fe: high pressure study of an Y2Fe17 single crystal.

In this Letter we present direct observation of the Fe helimagnetism in an Y2Fe17 single crystal under pressure. Combined neutron diffraction and magnetization measurements under pressure showed that the collinear ferromagnetic phase of Y2Fe17 is substituted by the pressure induced helical incommensurate phases. The complex pressure-temperature-field behavior of the pressure induced helical magnetic phases is attributed to intrinsic properties of the iron sublattice that gives a valuable contribution to the discussion about dominating theoretical models of magnetism in gamma-Fe.

Pressure enhancement of the giant magnetocaloric effect in Tb5Si2Ge2.

Effects of temperature and pressure on magnetic, elastic, structural, and thermal properties of Tb5Si2Ge2 have been studied by means of macroscopic (thermal expansion and magnetization) and microscopic (neutron powder diffraction) techniques. We present evidence that the high-temperature second-order ferromagnetic transition can be coupled with the low-temperature first-order structural phase change into a single first-order magnetic-crystallographic transformation at and above a tricritical point in the vicinity of 8.6 kbar. This pressure-induced coupling has a remarkable effect on the magnetocaloric effect, transforming Tb5Si2Ge2 from an ordinary into a giant magnetocaloric effect material.

Pressure-induced three-dimensional ferromagnetic correlations in the giant magnetocaloric compound Gd5Ge4.

The giant magnetocaloric compound Gd5Ge4 is the only member of the Gd5(SixGe1-x)4 family where three-dimensional exchange interactions between two-dimensional correlated layers of the crystallographic structure are so weak that spontaneous ferromagnetism does not set in at any temperature. In this Letter we explore the possibility to reach the ferromagnetic state by application of hydrostatic pressure. Linear thermal expansion and magnetic measurements under pressure reveal that the reduction of the unit cell volume induces a spatially phase-segregated ground state below 10 kbar.

Pressure-enhanced 3D antiferromagnetic correlations in La(1.4)Sr(1.6)Mn(2)O(7).

Pressure effects on the stability of magnetic phases in La(1.4)Sr(1.6)Mn(2)O(7) have been studied using magnetization measurements and neutron diffraction. At ambient conditions this material is a quasi-two-dimensional ferromagnet. On cooling it becomes ordered three dimensionally: at 90 K La(1.4)Sr(1.6)Mn(2)O(7) it becomes an antiferromagnet, and at 65 K it undergoes a transition into a ferromagnetic phase. Using neutron diffraction techniques on a single crystal of La(1.4)Sr(1.6)Mn(2)O(7) it has been shown that these two magnetic phases belong to a single structural phase and do not coexist at low temperatures. The application of pressure enhances the antiferromagnetic correlations between the Mn(2)O(9) bilayers.

Optically active aromatic amino acids. Part VI. Synthesis and properties of (Leu5)-enkephalin analogues containing O-methyl-L-tyrosine1 with ring substitution at position 3'.

Twelve new [Tyr(Me)1, Leu5]-enkephalin analogues with substituents at position 3' of the Tyr ring have been synthesized using traditional solution methods. The substituents were -CO2H, -CONH2, -CO2Me, -(E)-CH=NOH, -(E)-CH=NOMe and CH2OH. The analogues were C-terminated with methyl esters, amides or as free acids. In the in vitro biological assays a remarkable agonist activity to the opiate receptor mu in guinea pig ileum (GPI) relative to Leu-ENK was shown by the following: Leu-ENK, 100; [Tyr(Me)(3'-CO2Me)1, Leu-OMe5]-ENK (I), 8.1; [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OMe5]-ENK (VI), 26.2; [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OH5]-ENK (VII), 2.9; [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-NH2(5)]-ENK (VIII), 4.7; and [Tyr(Me)(3'-CH2OH)1, Leu-OMe5]-ENK (X), 5.6. The agonist effect was naltrexone- or naloxone-reversible. The masking of the hydroxyl group in (E)-hydroxyiminomethyl group of analogue (VI) by O-methylation has totally abolished its GPI agonist activity. It seems that the (E)-CH=NOH group shows affinity and plays an analogous role to the phenol group Tyr1 in leucine-enkephalin and in the tyramine group of the opiate alkaloids. The analogues: [Tyr(Me)(3'-CO2Me)1, Leu-OMe5]-ENK (I), [Tyr(Me)(3'-CO2H)1, Leu-OMe5]-ENK (II), [Tyr(Me)(3'-CO2Me)1, Leu-NH2(5)]-ENK (III), [Tyr(Me)(3'-CO2H)1, Leu-NH2(5)]-ENK (IV), [Tyr(Me)(3'-CONH2)1, Leu-NH2(5)]-ENK (V), [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OMe5]-ENK (VI), [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OH5]-ENK (VII), [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-NH2(5)]-ENK (VIII), [Tyr(Me)(3'-(E)-CH=NOMe)1, Leu-OMe5]-ENK (IX), [Tyr(Me)(3'-CH2OH)1, Leu-OMe5]-ENK (X), [Tyr(Me)(3'-CH2OH)1, Leu-OH5]-ENK (XI) and [Tyr(Me)(3'-CH2OH)1, Leu-NH2(5)]-ENK (XII) under testing had no significant agonist activity to the enkephalinergic receptor in mouse vas deferens (MVD). All methyl esters of synthesized analogues of [Leu5]-ENK showed higher activity to mu receptors than structurally identical C-terminal amides. It is a surprising result since usually C-terminate amides are stronger agonists than C-terminate esters.