ZBTB2 links p53 deficiency to HIF-1-mediated hypoxia signaling to promote cancer aggressiveness.

Aberrant activation of the hypoxia-inducible transcription factor HIF-1 and dysfunction of the tumor suppressor p53 have been reported to induce malignant phenotypes and therapy resistance of cancers. However, their mechanistic and functional relationship remains largely unknown. Here, we reveal a mechanism by which p53 deficiency triggers the activation of HIF-1-dependent hypoxia signaling and identify zinc finger and BTB domain-containing protein 2 (ZBTB2) as an important mediator. ZBTB2 forms homodimers via its N-terminus region and increases the transactivation activity of HIF-1 only when functional p53 is absent. The ZBTB2 homodimer facilitates invasion, distant metastasis, and growth of p53-deficient, but not p53-proficient, cancers. The intratumoral expression levels of ZBTB2 are associated with poor prognosis in lung cancer patients. ZBTB2 N-terminus-mimetic polypeptides competitively inhibit ZBTB2 homodimerization and significantly suppress the ZBTB2-HIF-1 axis, leading to antitumor effects. Our data reveal an important link between aberrant activation of hypoxia signaling and loss of a tumor suppressor and provide a rationale for targeting a key mediator, ZBTB2, to suppress cancer aggressiveness.

Modulation of Electron Transfer Branches by Atrazine and Triazine Herbicides in Photosynthetic Reaction Centers.

Quinone analogue molecules, functioning as herbicides, bind to the secondary quinone site, QB, in type-II photosynthetic reaction centers, including those from purple bacteria (PbRC). Here, we investigated the impact of herbicide binding on electron transfer branches, using herbicide-bound PbRC crystal structures and employing the linear Poisson-Boltzmann equation. In contrast to urea and phenolic herbicides [Fufezan, C. Biochemistry 2005, 44, 12780-12789], binding of atrazine and triazine did not cause significant changes in the redox-potential (Em) values of the primary quinone (QA) in these crystal structures. However, a slight Em difference at the bacteriopheophytin in the electron transfer inactive branch (HM) was observed between the S(-)- and R(+)-triazine-bound PbRC structures. This discrepancy is linked to variations in the protonation pattern of the tightly coupled Glu-L212 and Glu-H177 pairs, crucial components of the proton uptake pathway in native PbRC. These findings suggest the existence of a QB-mediated link between the electron transfer inactive HM and the proton uptake pathway in PbRCs.

Energetics of the H-Bond Network in Exiguobacterium sibiricum Rhodopsin.

Exiguobacterium sibiricum rhodopsin (ESR) functions as a light-driven proton pump utilizing Lys96 for proton uptake and maintaining its activity over a wide pH range. Using a combination of methodologies including the linear Poisson-Boltzmann equation and a quantum mechanical/molecular mechanical approach with a polarizable continuum model, we explore the microscopic mechanisms underlying its pumping activity. Lys96, the primary proton uptake site, remains deprotonated owing to the loss of solvation in the ESR protein environment. Asp85, serving as a proton acceptor group for Lys96, does not form a low-barrier H-bond with His57. Instead, deprotonated Asp85 forms a salt-bridge with protonated His57, and the proton is predominantly located at the His57 moiety. Glu214, the only acidic residue at the end of the H-bond network exhibits a pKa value of ∼6, slightly elevated due to solvation loss. It seems likely that the H-bond network [Asp85···His57···H2O···Glu214] serves as a proton-conducting pathway toward the protein bulk surface.

Mechanism of the formation of proton transfer pathways in photosynthetic reaction centers.

In photosynthetic reaction centers from purple bacteria (PbRCs) from Rhodobacter sphaeroides, the secondary quinone QB accepts two electrons and two protons via electron-coupled proton transfer (PT). Here, we identify PT pathways that proceed toward the QB binding site, using a quantum mechanical/molecular mechanical approach. As the first electron is transferred to QB, the formation of the Grotthuss-like pre-PT H-bond network is observed along Asp-L213, Ser-L223, and the distal QB carbonyl O site. As the second electron is transferred, the formation of a low-barrier H-bond is observed between His-L190 at Fe and the proximal QB carbonyl O site, which facilitates the second PT. As QBH2 leaves PbRC, a chain of water molecules connects protonated Glu-L212 and deprotonated His-L190 forms, which serves as a pathway for the His-L190 reprotonation. The findings of the second pathway, which does not involve Glu-L212, and the third pathway, which proceeds from Glu-L212 to His-L190, provide a mechanism for PT commonly used among PbRCs.

Carcinogenesis and epidemiology of cervical cancer: The hallmark of human papillomavirus-associated cancer.

Cervical cancer affects women worldwide and is the most common human papillomavirus (HPV)-associated cancer. Carcinogenesis caused by HPV results in specific cancer behavior because of the underlying viral infection. The mechanism and timing of the transformation from viral infection to cancer cells have been elucidated in detail. Treatments for this cancer are based on its characteristics and are being implemented. Moreover, HPV infection is widespread worldwide and is transmitted through sexual activity. Although the HPV vaccination is the most effective strategy of preventing cervical cancer, it is not feasible to vaccinate the entire human population especially in low- and middle-income countries. In order to consider the next step for HPV vaccination, we need to understand the characteristics of HPV carcinogenesis and cervical cancer. Additionally, treatment aimed at preservation of reproductive function in patients with cervical cancer is often required, as the cervix is a reproductive organ and because the disease is more prevalent in the adolescent and young adult generation. Thus, there are still many challenges in the diagnosis, treatment, and prevention of cervical cancer.

FOUR-WEEK ORAL ADMINISTRATION OF BALOXAVIR MARBOXIL AS AN ANTI-INFLUENZA VIRUS DRUG SHOWS NO TOXICITY IN CHICKENS.

High pathogenicity avian influenza is an acute zoonotic disease with high mortality in birds caused by a high pathogenicity avian influenza virus (HPAIV). Recently, HPAIV has rapidly spread worldwide and has killed many wild birds, including endangered species. Baloxavir marboxil (BXM), an anti-influenza agent used for humans, was reported to reduce mortality and virus secretion from HPAIV-infected chickens (Gallus domesticus, order Galliformes) at a dosage of ≥2.5 mg/kg when administered simultaneously with viral challenge. Application of this treatment to endangered birds requires further information on potential avian-specific toxicity caused by repeated exposure to BXM over the long term. To obtain information of potential avian-specific toxicity, a 4-wk oral repeated-dose study of BXM was conducted in chickens (n = 6 or 7 per group), which are commonly used as laboratory avian species. The study was conducted in reference to the human pharmaceutical guidelines for nonclinical repeated-dose drug toxicity studies to evaluate systemic toxicity and exposure. No adverse changes were observed in any organs examined, and dose proportional increases in systemic exposure to active pharmaceutical ingredients were noted from 12.5 to 62.5 mg/kg per day. BXM showed no toxicity to chickens at doses of up to 62.5 mg/kg per day, at which systemic exposure was approximately 71 times higher than systemic exposure at 2.5 mg/kg, the reported efficacious dosage amount, in HPAIV-infected chickens. These results also suggest that BXM could be considered safe for treating HPAIV-infected endangered birds due to its high safety margin compared with the efficacy dose. The data in this study could contribute to the preservation of endangered birds by using BXM as a means of protecting biodiversity.

Stretching vibrational frequencies and pKa differences in H-bond networks of protein environments.

The experimentally measured stretching vibrational frequencies of O-D [νO-D(donor)] and C=O [νC=O(donor)] H-bond donor groups can provide valuable information about the H-bonds in proteins. Here, using a quantum mechanical/molecular mechanical approach, the relationship between these vibrational frequencies and the difference in pKa values between H-bond donor and acceptor groups [ΔpKa(donor … acceptor)] in bacteriorhodopsin and photoactive yellow protein environments was investigated. The results show that νO-D(donor) is correlated with ΔpKa(donor … acceptor), regardless of the specific protein environment. νC=O(donor) is also correlated with ΔpKa(donor … acceptor), although the correlation is weak because the C=O bond does not have a proton. Importantly, the shifts in νO-D(donor) and νC=O(donor) are not caused by changes in pKa(donor) alone, but rather by changes in ΔpKa(donor … acceptor). Specifically, a decrease in ΔpKa(donor … acceptor) can lead to proton release from the H-bond donor group toward the acceptor group, resulting in shifts in the vibrational frequencies of the protein environment. These findings suggest that changes in the stretching vibrational frequencies, in particular νO-D(donor), can be used to monitor proton transfer in protein environments.

Quantum mechanical analysis of excitation energy transfer couplings in photosystem II.

We evaluated excitation energy transfer (EET) coupling (J) between all pairs of chlorophylls (Chls) and pheophytins (Pheos) in the protein environment of photosystem II based on the time-dependent density functional theory with a quantum mechanical/molecular mechanics approach. In the reaction center, the EET coupling between Chls PD1 and PD2 is weaker (|J(PD1/PD2)| = 79 cm-1), irrespective of a short edge-to-edge distance of 3.6 Å (Mg-to-Mg distance of 8.1 Å), than the couplings between PD1 and the accessory ChlD1 (|J(PD1/ChlD2)| = 104 cm-1) and between PD2 and ChlD2 (|J(PD2/ChlD1)| = 101 cm-1), suggesting that PD1 and PD2 are two monomeric Chls rather than a "special pair". There exist strongly coupled Chl pairs (|J| > âˆ¼100 cm-1) in the CP47 and CP43 core antennas, which may be candidates for the red-shifted Chls observed in spectroscopic studies. In CP47 and CP43, Chls ligated to CP47-His26 and CP43-His56, which are located in the middle layer of the thylakoid membrane, play a role in the "hub" that mediates the EET from the lumenal to stromal layers. In the stromal layer, Chls ligated to CP47-His466, CP43-His441, and CP43-His444 mediate the EET from CP47 to ChlD2/PheoD2 and from CP43 to ChlD1/PheoD1 in the reaction center. Thus, the excitation energy from both CP47 and CP43 can always be utilized for the charge-separation reaction in the reaction center.

pH-Dependent Binding and Releasing Mechanism of Acetate in the Inner Water Cavity of Heliorhodopsin.

The high-resolution structure of heliorhodopsin crystallized at low pH reveals the presence of a planar triangle molecule, acetate, in the inner water cavity. Here, we investigate how the acetate molecule is stabilized at the counterion Glu107 moiety, using molecular dynamics (MD) simulations and a quantum mechanical/molecular mechanical (QM/MM) approach. QM/MM calculations indicate that the density is best described as acetate among triangle acids, including nitric acid and bicarbonate. The calculated protonation state indicates that protonated acetate donates an H-bond to deprotonated Glu107 in the low-pH crystal structure. The observed red-shift of ∼30 nm in the absorption wavelength with pKa ≈ 4 is likely due to the His23/His80 protonation, rather than the Glu107 protonation. MD simulations also show that acetate can exist at the Glu107 moiety only when it is protonated. When ionized, acetate is released from the Glu107 moiety via Asn101 at the channel bottleneck and Arg91 on the intracellular protein surface. These observations could explain how acetate binds at low pH and releases at high pH.

Substitution of Ca2+ and changes in the H-bond network near the oxygen-evolving complex of photosystem II.

Ca2+, which provides binding sites for ligand water molecules W3 and W4 in the Mn4CaO5 cluster, is a prerequisite for O2 evolution in photosystem II (PSII). We report structural changes in the H-bond network and the catalytic cluster itself upon the replacement of Ca2+ with other alkaline earth metals, using a quantum mechanical/molecular mechanical approach. The small radius of Mg2+ makes W3 donate an H-bond to D1-Glu189 in Mg2+-PSII. If an additional water molecule binds at the large surface of Ba2+, it donates H-bonds to D1-Glu189 and the ligand water molecule at the dangling Mn, altering the H-bond network. The potential energy profiles of the H-bond between D1-Tyr161 (TyrZ) and D1-His190 and the interconversion between the open- and closed-cubane S2 conformations remain substantially unaltered upon the replacement of Ca2+. Remarkably, the O5⋯Ca2+ distance is shortest among all O5⋯metal distances irrespective of the radius being larger than that of Mg2+. Furthermore, Ca2+ is the only alkaline earth metal that equalizes the O5⋯metal and O2⋯metal distances and facilitates the formation of the symmetric cubane structure.

Acquirement of water-splitting ability and alteration of the charge-separation mechanism in photosynthetic reaction centers.

In photosynthetic reaction centers from purple bacteria (PbRC) and the water-oxidizing enzyme, photosystem II (PSII), charge separation occurs along one of the two symmetrical electron-transfer branches. Here we report the microscopic origin of the unidirectional charge separation, fully considering electron-hole interaction, electronic coupling of the pigments, and electrostatic interaction with the polarizable entire protein environments. The electronic coupling between the pair of bacteriochlorophylls is large in PbRC, forming a delocalized excited state with the lowest excitation energy (i.e., the special pair). The charge-separated state in the active branch is stabilized by uncharged polar residues in the transmembrane region and charged residues on the cytochrome c2 binding surface. In contrast, the accessory chlorophyll in the D1 protein (ChlD1) has the lowest excitation energy in PSII. The charge-separated state involves ChlD1•+ and is stabilized predominantly by charged residues near the Mn4CaO5 cluster and the proceeding proton-transfer pathway. It seems likely that the acquirement of water-splitting ability makes ChlD1 the initial electron donor in PSII.

Conformational Changes and H-Bond Rearrangements during Quinone Release in Photosystem II.

In photosystem II (PSII) and photosynthetic reaction centers from purple bacteria (PbRC), the electron released from the electronically excited chlorophyll is transferred to the terminal electron acceptor quinone, QB. QB accepts two electrons and two protons before leaving the protein. We investigated the molecular mechanism of quinone exchange in PSII, conducting molecular dynamics (MD) simulations and quantum mechanical/molecular mechanical (QM/MM) calculations. MD simulations suggest that the release of QB leads to the transformation of the short helix (D1-Phe260 to D1-Ser264), which is adjacent to the stromal helix de (D1-Asn247 to D1-Ile259), into a loop and to the formation of a water-intake channel. Water molecules enter the QB binding pocket via the channel and form an H-bond network. QM/MM calculations indicate that the H-bond network serves as a proton-transfer pathway for the reprotonation of D1-His215, the proton donor during QBH-/QBH2 conversion. Together with the absence of the corresponding short helix but the presence of Glu-L212 in PbRC, it seems likely that the two type-II reaction centers undergo quinone exchange via different mechanisms.

Polygalae Radix shortens the circadian period through activation of the CaMKII pathway.

CONTEXT: The mammalian circadian clock system regulates physiological function. Crude drugs, containing Polygalae Radix, and Kampo, combining multiple crude drugs, have been used to treat various diseases, but few studies have focussed on the circadian clock. OBJECTIVE: We examine effective crude drugs, which cover at least one or two of Kampo, for the shortening effects on period length of clock gene expression rhythm, and reveal the mechanism of shortening effects. MATERIALS AND METHODS: We prepared 40 crude drugs. In the in vitro experiments, we used mouse embryonic fibroblasts from PERIOD2::LUCIFERASE knock-in mice (background; C57BL/6J mice) to evaluate the effect of crude drugs on the period length of core clock gene, Per2, expression rhythm by chronic treatment (six days) with distilled water or crude drugs (100 µg/mL). In the in vivo experiments, we evaluated the free-running period length of C57BL/6J mice fed AIN-93M or AIN-93M supplemented with 1% crude drug (6 weeks) that shortened the period length of the PERIOD2::LUCIFERASE expression rhythm in the in vitro experiments. RESULTS: We found that Polygalae Radix (ED50: 24.01 µg/mL) had the most shortened PERIOD2::LUCIFERASE rhythm period length in 40 crude drugs and that the CaMKII pathway was involved in this effect. Moreover, long-term feeding with AIN-93M+Polygalae Radix slightly shortened the free-running period of the mouse locomotor activity rhythm. DISCUSSION AND CONCLUSIONS: Our results indicate that Polygalae Radix may be regarded as a new therapy for circadian rhythm disorder and that the CaMKII pathway may be regarded as a target pathway for circadian rhythm disorders.

Role of redox-inactive metals in controlling the redox potential of heterometallic manganese-oxido clusters.

Photosystem II (PSII) contains Ca2+, which is essential to the oxygen-evolving activity of the catalytic Mn4CaO5 complex. Replacement of Ca2+ with other redox-inactive metals results in a loss/decrease of oxygen-evolving activity. To investigate the role of Ca2+ in this catalytic reaction, we investigate artificial Mn3[M]O2 clusters redox-inactive metals  [M] ([M] = Mg2+, Ca2+, Zn2+, Sr2+, and Y3+), which were synthesized by Tsui et al. (Nat Chem 5:293, 2013). The experimentally measured redox potentials (Em) of these clusters are best described by the energy of their highest occupied molecular orbitals. Quantum chemical calculations showed that the valence of metals predominantly affects Em(MnIII/IV), whereas the ionic radius of metals affects Em(MnIII/IV) only slightly.

Clinical utility of thoracoscopy in elderly tuberculous pleurisy patients under local anesthesia.

INTRODUCTION: Diagnosing tuberculous pleurisy is important in Japan because it currently has a moderate tuberculosis prevalence. However, physicians often have difficulty making a diagnosis. It was reported that thoracoscopy under local anesthesia is useful for the diagnosis of tuberculous pleurisy, but there are no reports focusing on elderly patients. METHODS: In this study, the usefulness of thoracoscopy under local anesthesia was evaluated in elderly patients. Among 170 patients who underwent thoracoscopy under local anesthesia at our hospital during 11 years from January 2008 to December 2018, those aged 75 years or older (n = 75) were investigated retrospectively. RESULTS: A total of 55 patients underwent thoracoscopy under local anesthesia for detailed examination of pleural effusion of unknown cause. Of these, 18 were diagnosed as tuberculous pleurisy. The median age was 82 years (range: 75-92 years). The diagnosis of tuberculous pleurisy was made in 11 patients in whom Mycobacterium tuberculosis was detected and in four patients whose pathological findings indicated epithelioid granuloma accompanied by caseous necrosis. Clinical diagnosis was made in the remaining three patients based on thoracoscopic findings of the pleural cavity and a high level of adenosine deaminase in pleural fluid. No serious complications attributable to the examination were observed in any patient. CONCLUSIONS: Thoracoscopy under local anesthesia was useful for the diagnosis of tuberculous pleurisy in elderly patients, with useful information being also obtained for the treatment of tuberculosis.

Molecular characterization of Trichomonas gypaetinii isolated from the upper alimentary tract of Steller's sea eagles (Haliaeetus pelagicus) and white-tailed sea eagles (Haliaeetus albicilla) in Hokkaido, Japan.

Recent phylogenetic and morphologic studies of Trichomonas spp. suggests that there are more than 3 species that infect the upper alimentary tract of wild birds, which include T. gallinae, T. stableri, and T. gypaetinii. In this study, investigations were conducted on the prevalence of trichomonads in the upper alimentary tract of 12 Steller's sea eagles (Haliaeetus pelagicus) and 18 white-tailed sea eagles (H. albicilla). All birds were rescued from the wild and kept at a rehabilitation facility in Hokkaido, Japan, for variable durations and did not show any symptoms of trichomonosis. The ITS1-5.8SrRNA-ITS2 (ITS) genomic region of Trichomonas spp. was detected from 29 samples by PCR, and flagellates were confirmed from 4 samples by culture. Morphologic observations and measurement recordings were conducted under a light microscope on trophozoites obtained from the cultured isolates. Genomic sequences of the ITS, 18S ribosomal RNA (18S rRNA), Fe-hydrogenase, and RNA polymerase II largest subunit (Rpb1) regions were determined by direct sequencing, and phylogenetic analyses were conducted with previously published sequences of Trichomonas spp. All isolates were concluded as T. gypaetinii based on morphologic and molecular characterizations of the ITS and 18S rRNA genes. This is the first study to isolate T. gypaetinii from Haliaeetus eagles and further provide novel sequences of the Fe-hydrogenase and Rpb1 genes of T. gypaetinii. Both genomic regions also confirmed that T. gypaetinii belong to independent clusters from other Trichomonas spp.

Energetics of Ionized Water Molecules in the H-Bond Network near the Ca2+ and Cl- Binding Sites in Photosystem II.

In photosystem II, water oxidation occurs at the oxygen-evolving complex (OEC). The presence of a hydronium ion (H3O+) was proposed at the Cl- binding site and Ca2+-depleted OEC. Using a quantum mechanical/molecular mechanical approach, we report the stability of H3O+ in the PSII protein environment. Neither release of the proton from ligand water molecule W2 at the OEC nor formation of H3O+ at Cl- is energetically favorable. In contrast, H3O+ can exist at the Ca2+-depleted OEC. Even when H3O+ exists in Ca2+-depleted PSII, the H-bond network of the redox-active tyrosine (TyrZ) remains unaltered, retaining the unusually short low-barrier H-bond with D1-His190, and the redox potential of TyrZ, Em(TyrZ), remains unaltered. These observations explain why the oxidation of the Ca2+-depleted Mn4O5 cluster by TyrZ (i.e., the S2 to S3 transition) is not inhibited at low pH. It seems likely that Ca2+ plays a role in not only (i) maintaining the H-bond network and facilitating TyrZ oxidation [tuning Em(TyrZ)] but also (ii) providing the valence of +2, decreasing the pKa of the ligand molecule (W1), and facilitating the release of the proton from W1 in the S2 to S3 transition together with Cl-.

Redox potentials along the redox-active low-barrier H-bonds in electron transfer pathways.

Low-barrier H-bonds form when the pKa values of the H-bond donor and acceptor moieties are nearly equal. Here, we report redox potential (Em) values along two redox-active low-barrier H-bonds in the water-oxidizing enzyme photosystem II (PSII), using a quantum mechanical/molecular mechanical approach. The low-barrier H-bond between D1-Tyr161 (TyrZ) and D1-His190 is located in the middle of the electron transfer pathway. When the proton is at D1-His190, Em(TyrZ) is the lowest and can serve as an electron donor to the oxidized chlorophyll PD1˙+. Em(TyrZ) and Em(D1-His190) are equal, and the TyrZD1-His190 pair serves as an electron acceptor to Mn4CaO5 when the proton is at TyrZ. In the low-barrier H-bond between D1-His215 and plastoquinone QB, located at the terminus of the electron transfer pathway, the driving force of electron transfer and electronic coupling between QA and QB are maximized when the proton arrives at QB. It seems likely that local proton transfer along redox-active low-barrier H-bonds can alter the driving force or electronic coupling for electron transfer.

Rigidly hydrogen-bonded water molecules facilitate proton transfer in photosystem II.

In the water-splitting enzyme photosystem II (PSII), the proton is released from the catalytic site and transferred to the protein bulk surface via the proton-relay mechanism. Proton transfer occurs in a proton-conducting channel consisting of a series of water molecules connected by hydrogen-bonded (H-bonded) chains. The water-transport protein aquaporin (AQP) also contains a water chain with structure similar to that of the PSII proton channel, although the water chain does not transport protons. We compared the PSII proton channel with the AQP water channel from the following standpoints: (1) the energetics of proton transfer based on crystal structures obtained from quantum mechanical/molecular mechanical calculations, and (2) fluctuations in water molecules obtained from molecular dynamics simulations. The results showed that residues facing the channel and acting as H-bonded partners of water molecules predominantly determined the proton-transfer ability. In PSII, the water chain is surrounded by H-bond acceptors (e.g., carbonyl groups), and the water chain transports protons where the water molecules are rigidly fixed. In AQP, the water chain is surrounded by hydrophobic sidechains or H-bond donors (e.g., NH2 groups), and it does not transport protons where the water molecules are flexible and fluctuating.

A case of invasive pulmonary aspergillosis diagnosed by transbronchial lung biopsy during treatment for diabetic ketoacidosis in a type 1 diabetic patient.

Invasive pulmonary aspergillosis (IPA) patients with non-hematological malignancy are far less than with hematological malignancy patients. We encountered a very rare case of IPA in which type 1 diabetes was the only conceivable risk factor. Further, according to the diagnostic categories of the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria for IPA, the frequency of proven diagnosis is very low. Here we report a proven IPA, which rapidly developed when the patient with type 1 diabetes was being treated for diabetic ketoacidosis, which was successfully treated with the combination therapy of voriconazole (VRCZ) and micafungin (MCFG), based on early diagnosis using bronchoscopy.