[Efficacy and influencing factors of immunotherapy combined with chemotherapy and bevacizumab in patients with non-small cell lung cancer after epidermal growth factor receptor tyrosine kinase inhibitors treatment failure].

Objective: To investigate the efficacy and influencing factors of immunotherapy combined with chemotherapy and bevacizumab in patients with non-small cell lung cancer (NSCLC) who failed epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) treatment. Methods: A retrospective analysis was made on the clinical data of 60 NSCLC patients who were treated with immunotherapy combined with chemotherapy and bevacizumab after EGFR-TKIs treatment failure in the Affiliated Cancer Hospital of Shandong First Medical University from January 2019 to March 2022. Patients were followed up by telephone or outpatient review up to October 1, 2022, with a median follow-up of 8.2 months (95%CI: 7.1-9.3). All 60 patients were followed up. The response evaluation criteria in solid tumors were used to evaluate the short-term efficacy. The adverse reactions of patients were evaluated according to the common terminology criteria for adverse events. The survival curve was drawn by Kaplan-Meier method. Cox proportional hazard regression models were utilized to analyze the influencing factors of progression-free survival (PFS). Results: Among the 60 NSCLC patients, 22 were males. The age ranged from 41 to 75 years, with a median age of 61 years. Eleven patients had partial response, 19 patients had stable disease and 30 patients had progressive disease. The median PFS was 8.2 months (95%CI: 7.2-9.2). The median PFS of patients with low expression of programmed death receptor-ligand 1 (PD-L1) [Tumor cell Proportion Score (TPS)<1%], moderate expression of PD-L1 (1%≤TPS≤49%), and high expression of PD-L1 (TPS≥50%) were 6.4 (95%CI: 4.8-8.0), 8.3 (95%CI: 7.3-9.3) and 10.6 months (95%CI: 7.2-14.1), respectively, and there were statistically significant differences (χ2=13.58, P<0.001). Multivariate Cox proportional risk regression model analysis showed that age>65 years old (HR=4.017, 95%CI: 1.468-10.992, P=0.007) was a risk factor for PFS in NSCLC patients who received immunotherapy combined with chemotherapy and bevacizumab after EGFR-TKIs treatment failure. Moderate expression of PD-L1 (HR=0.360, 95%CI: 0.139-0.930, P=0.035) and high expression of PD-L1 (HR=0.155, 95%CI: 0.039-0.625, P=0.009) were protective factors for PFS. Most of the treatment-related adverse reactions in the whole group were grade 1-2, including bone marrow suppression (n=24), nausea (n=25), decreased appetite (n=24), fatigue (n=22), vomiting (n=18), abnormal liver function (n=17), blood creatinine increased (n=10), and so on. These were tolerated by the patients. Conclusions: NSCLC patients who failed EGFR-TKIs treatment can tolerate adverse reactions related to immunotherapy combined with chemotherapy and bevacizumab treatment. PFS is significantly prolonged in those aged≤65 years and those with moderate and high expression of PD-L1.

[Probe variables: a tool for identification of unmeasured confounders in an observational study].

There are usually unknown or unmeasured confounders in the observational study, which is a significant challenge in epidemiological causal association research. This paper presents a tool for identification and effect assessment of unknown/unmeasured confounders in observational studies: probe variables. It can be divided into three forms: exposure probe variable, outcome probe variable, and mediation probe variable. The first two types can identify unknown/unmeasured confounding factors and estimate their size of effect to reveal the real correlation between exposure and outcome. The mediation probe variable controls for "mediating factors" to identify unmeasured confounders between exposure and results. The most significant difficulty in this theory's practice is selecting and determining "probe variables." Improper probe variables may introduce unknown confounders, which may lead to false identification of unmeasured confounders. Probe variables can be recommended as a sensitivity analysis in observational studies to help readers truly understand the association between exposure and outcomes and to increase the strength of evidence in observational epidemiological studies.

Distribution of vasopressin, oxytocin and vasoactive intestinal polypeptide in the hypothalamus and extrahypothalamic regions of tree shrews.

Vasopressin (VP), oxytocin (OXT) and vasoactive intestinal polypeptide (VIP) in the brain modulate physiological and behavioral processes in many vertebrates. Day-active tree shrews, the closest relatives of primates, live singly or in pairs in territories that they defend vigorously against intruding conspecifics. However, anatomy concerning peptidergic neuron distribution in the tree shrew brain is less clear. Here, we examined the distribution of VP, OXT and VIP immunoreactivity in the hypothalamus and extrahypothalamic regions of tree shrews (Tupaia belangeri chinensis) using the immunohistochemical techniques. Most of VP and OXT immunoreactive (-ir) neurons were found in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus. In addition, VP-ir or OXT-ir neurons were scattered in the preoptic area, anterior hypothalamic areas, dorsomedial hypothalamic nucleus, stria terminalis, bed nucleus of the stria terminalis and medial amygdala. Interestingly, a high density of VP-ir fibers within the ventral lateral septum was observed in males but not in females. Both VP-ir and VIP-ir neurons were found in different subdivisions of the suprachiasmatic nucleus (SCN) with partial overlap. VIP-ir cells and fibers were also scattered in the cerebral cortex, anterior olfactory nucleus, amygdala and dentate gyrus of the hippocampus. These findings provide a comprehensive description of VIP and a detailed mapping of VP and OXT in the hypothalamus and extrahypothalamic regions of tree shrews, which is an anatomical basis for the participation of these neuropeptides in the regulation of circadian behavior and social behavior.

Increased atypical PKC expression and activity in the phrenic motor nucleus following cervical spinal injury.

Atypical protein kinase C (aPKC) isoforms are expressed in phrenic motor neurons, a group of motor neurons critical for breathing. Following C2 cervical hemisection (C2HS), spontaneous plasticity occurs in crossed-spinal synaptic pathways to phrenic motor neurons, at least partially restoring inspiratory phrenic activity below the injury. Since aPKCs are necessary for synaptic plasticity in other systems, we tested the hypothesis that C2HS increases aPKC expression and activity in spinal regions associated with the phrenic motor nucleus. C2 laminectomy (sham) or C2HS was performed on adult, male Lewis rats. Ventral spinal segments C3-5 were harvested 1, 3 or 28 days post-surgery, and prepared for aPKC enzyme activity assays and immunoblots. Ventral cervical aPKC activity was elevated 1 and 28, but not 3, days post-C2HS (1 day: 63% vs sham ipsilateral to injury; p<0.05; 28 day: 426% vs sham; p<0.05; no difference in ipsilateral vs contralateral response). Total PKCζ/ι protein expression was unchanged by C2HS, but total and phosphorylated PKMζ (constitutively active PKCζ isoform) increased ipsilateral to injury 28 days post-C2HS (p<0.05). Ipsilateral aPKC activity and expression were strongly correlated (r(2)=0.675, p<0.001). In a distinct group of rats, immunohistochemistry confirmed that aPKCs are expressed in neurons 28 days post-C2HS, including large, presumptive phrenic motor neurons; aPKCs were not detected in adjacent microglia (OX-42 positive cells) or astrocytes (GFAP positive cells). Changes in aPKC expression in the phrenic motor nucleus following C2HS suggests that aPKCs may contribute to functional recovery following cervical spinal injury.

CREB regulation of BK channel gene expression underlies rapid drug tolerance.

Pharmacodynamic tolerance is believed to involve homeostatic mechanisms initiated to restore normal neural function. Drosophila exposed to a sedating dose of an organic solvent, such as benzyl alcohol or ethanol, acquire tolerance to subsequent sedation by that solvent. The slo gene encodes BK-type Ca(2+)-activated K(+) channels and has been linked to alcohol- and organic solvent-induced behavioral tolerance in mice, Caenorhabditis elegans (C. elegans) and Drosophila. The cyclic AMP response element-binding (CREB) proteins are transcription factors that have been mechanistically linked to some behavioral changes associated with drug addiction. Here, we show that benzyl alcohol sedation alters expression of both dCREB-A and dCREB2-b genes to increase production of positively acting CREB isoforms and to reduce expression of negatively acting CREB variants. Using a CREB-responsive reporter gene, we show that benzyl alcohol sedation increases CREB-mediated transcription. Chromatin immunoprecipitation assays show that the binding of dCREB2, with a phosphorylated kinase-inducible domain, increases immediately after benzyl alcohol sedation within the slo promoter region. Most importantly, we show that a loss-of-function allele of dCREB2 eliminates drug-induced upregulation of slo expression and the production of benzyl alcohol tolerance. This unambiguously links dCREB2 transcription factors to these two benzyl alcohol-induced phenotypes. These findings suggest that CREB positively regulates the expression of slo-encoded BK-type Ca(2+)-activated K(+) channels and that this gives rise to behavioral tolerance to benzyl alcohol sedation.

A non-circadian role for cAMP signaling and CREB activity in Drosophila rest homeostasis.

In the fruit fly, Drosophila melanogaster, rest shares features with mammalian sleep, including prolonged immobility, decreased sensory responsiveness and a homeostatic rebound after deprivation. To understand the molecular regulation of sleep-like rest, we investigated the involvement of a candidate gene, cAMP response-element binding protein (CREB). The duration of rest was inversely related to cAMP signaling and CREB activity. Acutely blocking CREB activity in transgenic flies did not affect the clock, but increased rest rebound. CREB mutants also had a prolonged and increased homeostatic rebound. In wild types, in vivo CREB activity increased after rest deprivation and remained elevated for a 72-hour recovery period. These data indicate that cAMP signaling has a non-circadian role in waking and rest homeostasis in Drosophila.

Tetracycline-inducible systems for Drosophila.

Since their inception, tetracycline (Tet)-inducible systems have become the method of choice for transgenic research. The Tet-Off systems have a number of advantages, including robust target induction using a relatively benign effector molecule. However, use of the Tet-On system has been fraught with difficulties, including high background expression in the absence of effector molecules and inconsistent gene induction. Recently, second generation Tet-On transactivators (TAs) have been described. In HeLa cells, they are far more efficient than the original reverse TA protein, and they exhibit lower background activity in the absence of effectors. Here we examine the most promising TA in transgenic Drosophila and characterize its in vivo properties. We report that low levels of doxycycline, when added to normal fly food, efficiently and rapidly induce target transgenes in adults, larvae, and embryos. This TA is superior to all other Tet-On proteins, and its performance is comparable to that of the widely used Tet-Off TA. In addition, combining the improved Tet-On TA with the Gal4-UAS (upstream-activating sequence) system produces robust, spatially restricted, temporally controlled transgene induction. Because this Tet-On TA is significantly more efficient than previous ones used in Drosophila, it is also possible to modulate gene induction by controlling the dosage of the antibiotic in the food.

Adaptable doxycycline-regulated gene expression systems for Drosophila.

We have engineered two new versions of the doxycycline (dox) inducible system for use in Drosophila. In the first system, we have used the ubiquitously expressed Drosophila actin5C promoter to express the Tet-Off transactivator (tTA) in all tissue. Induction of a luciferase target transgene begins 6 h after placing the flies on dox-free food. Feeding drug-free food to mothers results in universal target gene expression in their embryos. Larvae raised on regular food also show robust expression of a target reporter gene. In the second version, we have used the Gal4-UAS system to spatially limit expression of the transactivator. Dox withdrawal results in temporally- and spatially-restricted, inducible expression of luciferase in the adult head and embryo. Both the actin5C and Gal4-UAS versions produce more than 100-fold induction of luciferase in the adult, with virtually no leaky expression in the presence of drug. Reporter gene expression is also undetectable in larvae or embryos from mothers fed dox-containing food. Such tight control may be due to the incorporation of Drosophila insulator elements (SCS and SCS') into the transgenic vectors. These systems offer a practical, effective alternative to currently available expression systems in the Drosophila research community.

Location, location, location: the many addresses of memory formation.

Memory formation, like real estate, can be summarized succinctly-location, location, location. It is an emergent property involving different anatomical regions in the brain, sets of neuronal circuits, and cellular and molecular interactions between and within those neurons. At each of these levels of description, location continues to be a major organizing principle guiding researchers. The difficulty in the field is the integration of information between the various levels of analyses, and it is proposed that molecular reporters may help to fill that void.

The Drosophila dCREB2 gene affects the circadian clock.

We report the role of dCREB2, the Drosophila homolog of CREB/CREM, in circadian rhythms. dCREB2 activity cycles with a 24 hr rhythm in flies, both in a light:dark cycle and in constant darkness. A mutation in dCREB2 shortens circadian locomotor rhythm in flies and dampens the oscillation of period, a known clock gene. Cycling dCREB2 activity is abolished in a period mutant, indicating that dCREB2 and Period affect each other and suggesting that the two genes participate in the same regulatory feedback loop. We propose that dCREB2 supports cycling of the Period/Timeless oscillator. These findings support CREB's role in mediating adaptive behavioral responses to a variey of environmental stimuli (stress, growth factors, drug addiction, circadian rhythms, and memory formation) in mammals and long-term memory formation and circadian rhythms in Drosophila.

Drosophila learning and memory: recent progress and new approaches.

The processes of learning and memory have traditionally been studied in large experimental organisms (Aplysia, mice, rats and humans), where well-characterized behaviors are easily tested. Although Drosophila is one of the most experimentally tractable organisms, it has only recently joined the others as a model organism for learning and memory. Drosophila behavior has been studied for over 20 years; however, most of the work in the learning and memory field has focused on initial learning, because establishing memory in Drosophila has not been as straightforward as in other organisms. A major recent advance in this field has been the development of a training protocol that induces long-term memory in files. This made possible experiments that implicated the Drosophila CREB gene as a critical component in the consolidation of long-term memory, and paves the way for future experiments utilizing the well developed tools in Drosophila. This review will briefly summarize what is known in the field of Drosophila learning and memory to date, and discuss why the unique aspects of this field make traditional approaches difficult and reward the use of alternative paths of experimentation.

CREB and the formation of long-term memory.

Cyclic AMP response element binding protein (CREB)-responsive transcription plays a central role in the formation of long-term memory in Drosophila, Aplysia and mice. Agents that disrupt the activity of CREB specifically block the formation of long-term memory, whereas agents that increase the amount or activity of the transcription factor accelerate the process. These results have led to the recent hypothesis that CREB is pivotal in the switch from short-term (protein synthesis independent) to long-term (protein synthesis dependent) memory.

A Drosophila CREB/CREM homolog encodes multiple isoforms, including a cyclic AMP-dependent protein kinase-responsive transcriptional activator and antagonist.

We have characterized a Drosophila gene that is a highly conserved homolog of the mammalian cyclic AMP (cAMP)-responsive transcription factors CREB and CREM. Uniquely among Drosophila genes characterized to date, it codes for a cAMP-responsive transcriptional activator. An alternatively spliced product of the same gene is a specific antagonist of cAMP-inducible transcription. Analysis of the splicing pattern of the gene suggests that the gene may be the predecessor of the mammalian CREB and CREM genes.

CREB as a memory modulator: induced expression of a dCREB2 activator isoform enhances long-term memory in Drosophila.

Genetic studies of memory formation in Drosophila have revealed that the formation of a protein synthesis-dependent long-term memory (LTM) requires multiple training sessions. LTM is blocked specifically by induced expression of a repressor isoform of the cAMP-responsive element-binding protein (CREB). Here, we report an enhancement of LTM formation after induced expression of an activator isoform of dCREB2. Maximum LTM is achieved after one training session, and its formation depends on phosphorylation of the activator transgene. A model of LTM formation based on differential regulation of CREB isoforms is proposed.

The determinants of community health nursing in Taiwan: past, present, and future aspects.

There are many determinants that influence the development of community health nursing in Taiwan. Political reality and policy, economic development, social changes, universality of education, systematization of healthcare, and disease changes are among the main factors. The author individually describes these influences in reference to past and current situations. Then, the author discusses the future prospects of these influences on community health nursing in Taiwan.

Induction of a dominant negative CREB transgene specifically blocks long-term memory in Drosophila.

Consolidated memory after olfactory learning in Drosophila consists of two components, a cycloheximide-sensitive, long-term memory (LTM) and a cycloheximide-insensitive, anesthesia-resistant memory (ARM). Using an inducible transgene that expresses a dominant negative member of the fly CREB family, LTM was specifically and completely blocked only after induction, while ARM and learning were unaffected. These results suggest that LTM formation requires de novo gene expression probably mediated by CREB family genes.

p2 and inhibition of Tn5 transposition.

Mutations of Tn5 which decreased the amount of the shorter element-encoded protein (p2) were made. One mutation was a change in the translation initiation codon of the protein, while two other mutations were changes in the promoter of the transcript (T2) which codes for p2. Analysis of all three mutants indicates that they decreased the inhibition of transposition that the protein exerts (in trans) on another element. The mutants have complicated transposition behaviors. Analysis of the RNA and proteins synthesized from the mutants led to the proposal that p2 can inhibit transposition at normal physiological concentrations. Therefore p2 synthesized from a given element is partly responsible for controlling the transposition frequency of the element. The mutants also show that p1 is the only Tn5-encoded protein necessary for transposition.

Effect of dam methylation on Tn5 transposition.

The effect of dam methylation on Tn5 transposition was investigated by analyses of mutations in the host (Escherichia coli) and the element. Wild-type elements transposed at a higher frequency and showed higher levels of transposase expression in a dam-host. Mutations were made in the promoter region of the transcript that codes for the transposase. Transposition and transposase levels from these mutants were independent of the host methylation system. Measurements of the amount of RNA support the hypothesis that dam methylation exerts its effect on Tn5 transposition by modulating the frequency of transcriptional initiation of the transposase gene. Since Tn5 transposition increases when the transposase levels increase, at normal concentrations the amount of transposase is a rate-limiting factor that determines the transposition frequency of Tn5. Transposition of IS50, one of the insertion sequences that constitutes Tn5, is also sensitive to dam methylation by a second mechanism in addition to that of modulating transcriptional initiation. dam methylation, either directly or indirectly, inhibits the usage of IS50 sequences by the transposase. Thus, dam methylation can affect both the expression of the transposase and the DNA substrate upon which it acts.

dnaA, an essential host gene, and Tn5 transposition.

Mutations in dnaA, an essential gene in Escherichia coli, decrease the frequency of transposition of Tn5. An insertion mutation in the dnaA gene does not affect Tn5 gene expression. Therefore, the DnaA protein plays a role either in the transposition reaction itself or in some type of cellular regulation of transposition. Analysis of a mutation in the DnaA box, found at the outside end of IS50, is consistent with a direct interaction of the protein through these bases. IS50 transposition, which utilizes only one end containing a DnaA box, is not affected by dnaA mutations. Overproduction of the DnaA protein does not increase transposition frequencies in wild-type cells, even when the transposase is also overproduced.