The role of neural connexins in HeLa cell mobility and intercellular communication through tunneling tubes.

BACKGROUND: Membranous tunneling tubes (TTs) are a recently discovered new form of communication between remote cells allowing their electrical synchronization, migration, and transfer of cellular materials. TTs have been identified in the brain and share similarities with neuronal processes. TTs can be open-ended, close-ended or contain functional gap junctions at the membrane interface. Gap junctions are formed of two unapposed hemichannels composed of six connexin (Cx) subunits. There are evidences that Cxs also play channel-independent role in cell adhesion, migration, division, differentiation, formation of neuronal networks and tumorigenicity. These properties of Cxs and TTs may synergetically determine the cellular and intercellular processes. Therefore, we examined the impact of Cxs expressed in the nervous system (Cx36, Cx40, Cx43, Cx45, and Cx47) on: 1) cell mobility; 2) formation and properties of TTs; and 3) transfer of siRNA between remote cells through TTs. RESULTS: We have identified two types of TTs between HeLa cells: F-actin rich only and containing F-actin and α-tubulin. The morphology of TTs was not influenced by expression of examined connexins; however, Cx36-EGFP-expressing cells formed more TTs while cells expressing Cx43-EGFP, Cx45, and Cx47 formed fewer TTs between each other compared with wt and Cx40-CFP-expressing cells. Also, Cx36-EGFP and Cx40-CFP-expressing HeLa cells were more mobile compared with wt and other Cxs-expressing cells. TTs containing Cx40-CFP, Cx43-EGFP, or Cx47 gap junctions were capable of transmitting double-stranded small interfering RNA; however, Cx36-EGFP and Cx45 were not permeable to it. In addition, we show that Cx43-EGFP-expressing HeLa cells and laryngeal squamous cell carcinoma cells can couple to the mesenchymal stem cells through TTs. CONCLUSIONS: Different Cxs may modulate the mobility of cells and formation of TTs in an opposite manner; siRNA transfer through the GJ-containing TTs is Cx isoform-dependent.

Women's experiences of childbirth during the COVID-19 pandemic in Lithuania.

INTRODUCTION: Childbirth experiences depend on environmental factors, the provision of qualified medical and non-medical care, and the woman's psychological well-being. Stress experienced during pregnancy and childbirth affects a woman's psychological well-being. The aim of this study was to determine the care of women who gave birth during the COVID-19 pandemic in Lithuania. METHODS: This qualitative study used an interview method to reveal women's childbirth experiences during and before the pandemic in Lithuania. The data obtained during the interview were analyzed using qualitative content analysis. Interviews were taken from 15 women who gave birth at least twice, i.e. the first time until March 2020 (but not earlier than March 2019) and gave birth again during the COVID-19 pandemic (March 2020 - January 2021). RESULTS: A total of 15 women participanted in the interviews. The experience of childbirth before the COVID-19 pandemic was seen as largely positive by women, but childbirth during the COVID-19 pandemic was mentioned as more complex and negative because of the challenges posed by the pandemic, but easier for other reasons not affected by the pandemic. The results of our study show that a higher proportion of women were satisfied with delivery care in hospitals, were happy, and praised the work of midwives and other staff, which mainly contributed to a positive experience. CONCLUSIONS: The COVID-19 pandemic posed particular challenges to women's childbirth experiences, and not enough attention was paid to mental health. The stress that was exacerbated during the pandemic period had a profound impact on the pregnant woman in Lithuania.

Care of women and application of the principle of informed consent to interventions during birth in the COVID-19 pandemic period.

INTRODUCTION: In the early phases of the COVID-19 pandemic, inadequate intrapartum care protocols were in place. Many organizations have responded promptly and recognized the importance of adherence to appropriate guidelines. The International Confederation of Midwives issued an official statement on 29 March 2020, which states that every woman has the right to information, to give consent, to refuse consent, and to have her choices and decisions respected and upheld. No research has been conducted in Lithuania to reveal the care of women who gave birth during the COVID-19 pandemic and the application of informed consent to interventions. METHODS: This study is quantitative of cross-sectional design. An anonymous questionnaire survey method was used. One hundred fifty-two women who gave birth in Lithuania during the COVID-19 pandemic (March 2020 - May 2022) and had COVID-19 infection during childbirth, participated in the study. Statistical data analysis was performed. RESULTS: During the COVID-19 pandemic, women's care was characterized by always or almost always adequate information from health professionals on all issues to minimize the stress of new procedures necessitated by the COVID-19 pandemic and allowing them to stay with newborns as long as possible. The application of the principle of informed consent to interventions during the COVID-19 pandemic was not always applied to the performance of transvaginal examination manual compression of the uterine fundus to facilitate the expulsion period. CONCLUSIONS: Most women said that they were properly informed by healthcare professionals about all questions related to the new procedures that became necessary due to the COVID-19 pandemic and felt included in their own choice. However, mothers felt the need of relatives during childbirth, and consent was often not asked for vaginal examination.

Modulation of Connexin-36 Gap Junction Channels by Intracellular pH and Magnesium Ions.

Connexin-36 (Cx36) protein forms gap junction (GJ) channels in pancreatic beta cells and is also the main Cx isoform forming electrical synapses in the adult mammalian brain. Cx36 GJs can be regulated by intracellular pH (pHi) and cytosolic magnesium ion concentration ([Mg2+]i), which can vary significantly under various physiological and pathological conditions. However, the combined effect and relationship of these two factors over Cx36-dependent coupling have not been previously studied in detail. Our experimental results in HeLa cells expressing Cx36 show that changes in both pHi and [Mg2+]i affect junctional conductance (gj) in an interdependent manner; in other words, intracellular acidification cause increase or decay in gj depending on whether [Mg2+]i is high or low, respectively, and intracellular alkalization cause reduction in gj independently of [Mg2+]i. Our experimental and modelling data support the hypothesis that Cx36 GJ channels contain two separate gating mechanisms, and both are differentially sensitive to changes in pHi and [Mg2+]i. Using recombinant Cx36 we found that two glutamate residues in the N-terminus could be partly responsible for the observed interrelated effect of pHi and [Mg2+]i. Mutation of glutamate at position 8 attenuated the stimulatory effect of intracellular acidification at high [Mg2+]i, while mutation at position 12 and double mutation at both positions reversed stimulatory effect to inhibition. Moreover, Cx36*E8Q lost the initial increase of gj at low [Mg2+]i and double mutation lost the sensitivity to high [Mg2+]i. These results suggest that E8 and E12 are involved in regulation of Cx36 GJ channels by Mg2+ and H+ ions.

Molecular basis for potentiation of Cx36 gap junction channel conductance by n-alcohols and general anesthetics.

In our recent study, we have demonstrated that short carbon chain n-alcohols (up to octanol) stimulated while long carbon chain n-alcohols inhibited the conductance of connexin (Cx) 36 (Cx36) gap junction (GJ) channels. In contrast, GJ channels composed of other types of Cxs all were inhibited by n-alcohols independent of their carbon chain length. To identify the putative structural domains of Cx36, responsible for the dual effect of n-alcohols, we performed structural modeling of Cx36 protein docking with hexanol and isoflurane that stimulated as well as nonanol and carbenoxolone that inhibited the conductance of Cx36 GJs and revealed their multiple common docking sites and a single pocket accessible only to hexanol and isoflurane. The pocket is located in the vicinity of three unique cysteine residues, namely C264 in the fourth, and C92 and C87 in the second transmembrane domain of the neighboring Cx36 subunits. To examine the hypothesis that disulphide bonding might be involved in the stimulatory effect of hexanol and isoflurane, we generated cysteine substitutions in Cx36 and demonstrated by a dual whole-cell patch-clamp technique that in HeLa (human cervix carcinoma cell line) and N2A (mouse neuroblastoma cell line) cells these mutations reversed the stimulatory effect of hexanol and isoflurane to inhibitory one, typical of other Cxs that lack respective cysteines and a specific docking pocket for these compounds. Our findings suggest that the stimulatory effect of hexanol and isoflurane on Cx36 GJ conductance could be achieved by re-shuffling of the inter-subunit disulphide bond between C264 and C92 to the intra-subunit one between C264 and C87.