REVERCE: a randomized phase II study of regorafenib followed by cetuximab versus the reverse sequence for previously treated metastatic colorectal cancer patients.

BACKGROUND: The objective of this randomized phase II trial was to evaluate efficacy and safety of the therapeutic sequence of regorafenib followed by cetuximab, compared with cetuximab followed by regorafenib, as the current standard sequence for metastatic colorectal cancer patients. PATIENTS AND METHODS: Patients with KRAS exon 2 wild-type metastatic colorectal cancer after failure of fluoropyrimidine, oxaliplatin, and irinotecan were randomized to receive sequential treatment with regorafenib followed by cetuximab ± irinotecan (R-C arm), or the reverse sequence [cetuximab ± irinotecan followed by regorafenib (C-R arm)]. The primary end point was overall survival (OS). Key secondary end points included progression-free survival (PFS) with initial treatment (PFS1), PFS with second treatment (PFS2), safety, and quality of life. Exploratory end points included serial biomarker analyses, including oncogenic alterations from circulating tumor DNA or multiple serum or plasma proteins. RESULTS: One-hundred one patients were randomized and eligible for efficacy analysis. Sequential treatment was successful in 86% patients in both arms. Median OS for R-C and C-R was 17.4 and 11.6 months, respectively (P = 0.0293), with a hazard ratio (HR) of 0.61 for OS [95% confidence interval (CI) 0.39-0.96]. The HR for PFS1 (regorafenib in R-C versus cetuximab in C-R) was 0.97 (95% CI 0.61-1.54), and PFS2 (C in R-C versus R in C-R) was 0.29 (95% CI 0.17-0.50). No unexpected safety signals were observed. The quality of life scores during the entire treatment period was not significantly different between the two arms. Circulating biomarker analyses showed emerging oncogenic alterations in RAS, BRAF, EGFR, HER2, and MET, which were more commonly detected after cetuximab than after regorafenib. CONCLUSIONS: The therapeutic sequence of regorafenib followed by cetuximab suggests a longer OS than the current standard sequence.

Phase II study of trifluridine/tipiracil plus bevacizumab by RAS mutation status in patients with metastatic colorectal cancer refractory to standard therapies: JFMC51-1702-C7.

BACKGROUND: Although the efficacy of trifluridine/tipiracil (FTD/TPI) plus bevacizumab (BEV) against metastatic colorectal cancer (mCRC) has been demonstrated, little is known about its effectiveness upon disease stratification by RAS mutations. In this phase II study, we investigated the efficacy and safety profiles of FTD/TPI in mCRC according to RAS mutation status. PATIENTS AND METHODS: Eligible patients were mCRC refractory or intolerant to all standard therapies other than FTD/TPI and regorafenib. Patients received 4-week cycles of treatment with FTD/TPI (35 mg/m2, twice daily, days 1-5 and 8-12) and bevacizumab (5 mg/kg, days 1 and 15). The primary endpoint was disease control rate (DCR). The null hypothesis of DCR in both RAS wild-type (WT) and mutant (MUT) cohorts was 44%, assuming a one-sided significance level of 5.0%. The necessary sample size was estimated to be 49 patients (target sample size: 50 patients) for each cohort. RESULTS: Between January and September 2018, 102 patients were enrolled, and 97 patients fulfilled the eligibility criteria (48 in the RAS WT cohort and 49 in the RAS MUT cohort). DCRs in the RAS WT and MUT cohort were 66.7% [90% confidence interval (CI), 53.9%-77.8%, P = 0.0013] and 55.1% (90% CI, 42.4%-67.3%, P = 0.0780), respectively. The median progression-free survival (PFS) and overall survival (OS) were 3.8 and 9.3 months, respectively, in the RAS WT cohort and 3.5 and 8.4 months, respectively, in the RAS MUT cohort. The most common grade 3 or higher adverse event in both cohorts was neutropenia (46% in the RAS WT cohort and 62% in the RAS MUT cohort), without unexpected safety signals. CONCLUSIONS: FTD/TPI plus bevacizumab showed promising activity with an acceptable safety profile for pretreated mCRC, regardless of RAS mutation status, although the efficacy outcomes tended to be better in RAS WT.

An AU-box motif upstream of the SD sequence of light-dependent psbA transcripts confers mRNA instability in darkness in cyanobacteria.

The psbA2 gene of a unicellular cyanobacterium, Microcystis aeruginosa K-81, encodes a D1 protein homolog in the reaction center of photosynthetic Photosystem II. The expression of the psbA2 transcript has been shown to be light-dependent as assessed under light and dark (12/12 h) cycling conditions. We aligned the 5'-untranslated leader regions (UTRs) of psbAs from different photosynthetic organisms and identified a conserved sequence, UAAAUAAA or the 'AU-box', just upstream of the SD sequences. To clarify the role of 5'-upstream cis-elements containing the AU-box for light-dependent expression of psbA2, a series of deletion and point mutations in the region were introduced into the genome of heterologous cyanobacterium Synechococcus sp. strain PCC 7942, and psbA2 expression was examined. A clear pattern of light-dependent expression was observed in recombinant cyanobacteria carrying the K-81 psbA2 -38/+36 region (which includes the minimal promoter element and a light-dependent cis-element with the AU-box), +1 indicating the transcription start site. A constitutive pattern of expression, in which the transcripts remained almost stable under dark conditions, was obtained in cells harboring the -38/+14 region (the minimal element), indicating that the +14/+36 region with the AU-box is important for the observed light-dependent expression. Point mutations analyses within the AU-box also revealed that changes in number, direction and identity (as assayed by adenine/uridine nucleotide substitutions) influenced the light-dependent pattern of expression. The level of psbA2 transcripts increased markedly in CG- or deletion-box mutants in the dark, strongly indicating that the AU- (AT-) box acts as a negative cis-element. Furthermore, characterization of transcript accumulation in cells treated with rifampicin suggests that psbA2 5'-mRNA is unstable in the dark, supporting the view that the light-dependent expression is controlled at the post-transcriptional level. We discuss various mechanisms that may lead to altered mRNA stability such as the binding of factor(s) or ribosomes to the 5'-UTR and possible roles of the AU-box motif and the SD sequence.

Specific recognition of the cyanobacterial psbA promoter by RNA polymerases containing principal sigma factors.

The psbA2 gene of a unicellular cyanobacterium, Microcystis aeruginosa K-81, encodes a D1 protein homolog in the reaction center of photosynthetic Photosystem II. To clarify the promoter recognition by a sigma factor of RNA polymerase, in vivo and in vitro analyses were performed for the photosynthetic gene. Although the specific transcript from the psbA2 promoter, whose sequence is of Escherichia coli consensus type, was observed in both cyanobacterium K-81 and E. coli cells, the expression was light-dependent in K-81 whereas it was constitutive in E. coli under the conditions of light and darkness (L/D). The specific psbA2-dependent transcripts were also detected in vitro by RNA polymerases containing the principal sigma factors, E. coli sigma70 and K-81 sigmaA1 (constitutively exists in K-81 grown under L/D cycles). Furthermore, a series of promoter fragments were constructed to confirm minimal cis elements for the in vitro psbA2 transcription. A -80 to +6 or -38 to +46 region, the sequences of which consisted of a core promoter (-38 to +6), was identified as the potential minimal cis element using the RNA polymerase fraction (*EsigmaA1) containing sigmaA1 partially purified from K-81. These results suggest that the psbA2 transcription with the minimal sequence was induced by the RNA polymerase (EsigmaA1) containing the principal sigma factor, sigmaA1, under both light and dark conditions in K-81.

A novel genetic organization: the leuA-rpoD1 locus in the cyanobacterium Microcystis aeruginosa K-81.

We cloned and sequenced the region upstream of rpoD1, which encodes a principal sigma factor in the cyanobacterium Microcystis aeruginosa K-81. An open reading frame (orf1, 1599 bp) was discovered, the deduced amino-acid sequence of which (533 aa, 58, 016 Da) exhibits homology to another bacterial leuA gene product, 2-isopropylmalate synthase. The leuA (orf1) gene specifically complemented an E. coli leuA mutant. The 5'-upstream region of leuA did not contain possible leader peptide or stem-loop structures for attenuation. These findings indicate that the genetic structure of the leuA-rpoD1 locus in M. aeruginosa K-81 significantly differs from those of known leuA and rpoD loci found in other bacteria.

A new sigma factor homolog in a cyanobacterium: cloning, sequencing, and light-responsive transcripts of rpoD2 from Microcystis aeruginosa K-81.

We isolated an rpoD2 gene encoding the potential sigma factor of RNA polymerase from the cyanobacterium Microcystis aeruginosa K-81, which can perform photosynthesis. The deduced amino acid sequence of RpoD2 (sigmaA2) exhibits extensive homology to other eubacterial RpoD proteins. This gene possessed multiple 5'-end transcripts, expressed specifically under light (P(L)), dark (P(D)), or constitutively light/dark (P(C)) conditions during exponential cell growth.

Dimer form of phosphorylated Spo0A, a transcriptional regulator, stimulates the spo0F transcription at the initiation of sporulation in Bacillus subtilis.

The Spo0A protein of Bacillus subtilis is a transcriptional regulator that shows extensive homology to the regulator proteins in bacterial two-component regulatory systems. Phosphorylation of Spo0A is absolutely necessary for the initiation of sporulation. We now show that phospho-Spo0A is a dimer, binds specifically to the spo0F promoter region, and stimulates the transcription from the P2 promoter recognized by sigma H-RNA polymerase. Biochemical and biological analyses suggest that phospho-Spo0A interacts directly with the "0A-like box" sequence (TGTCGTA) located in the spo0F promoter region. Phosphorylation of Spo0A enhanced its affinity to the 0A-like box. Evidence is also presented that the spo0F promoter region contains a static bend having two sets of oligo(dA-dT) tracts. It was demonstrated that the bending region overlaps with the recognition site for the phospho-Spo0A.

Cloning, sequencing and characterization of the gene encoding a principal sigma factor homolog from the cyanobacterium Microcystis aeruginosa K-81.

We cloned and sequenced the rpoD1 gene of Microcystis aeruginosa K-81, a unicellular colony-forming cyanobacterium that can perform photosynthesis involving light-responsive gene expression. The deduced amino acid sequence of RpoD1 exhibited extensive homology to the other eubacterial principal sigma factors. Primer extension and Western blot analyses revealed that the rpoD1 gene, which encodes a principle sigma factor homolog, had two transcription start points, P1 and P2. These transcripts, and the corresponding protein, constitutively appeared in M. aeruginosa, irrespective of light or dark conditions.

Signal transduction and sporulation in Bacillus subtilis: heterologous phosphorylation of Spo0A, a sporulation initiation gene product.

Spo0A is both a positive and a negative transcriptional regulator which plays a very important role in sporulation initiation in Bacillus subtilis. Its N-terminal amino acid sequence is homologous to that of regulator proteins of the two-component regulatory systems involved in signal transduction in bacteria. Phosphorylation of Spo0A through phosphorelay has been reported by Burbulys et al. (1991). In this study, we found that (i) Spo0A is phosphorylated effectively with phospho-EnvZ* (N-terminal truncated EnvZ), which is a heterologous osmotic sensor protein in Escherichia coli, and (ii) a phosphorylation deficient mutant of Spo0A protein is completely defective in initiating sporulation. These results suggest that Spo0A phosphorylation may be an essential event in signal transduction of sporulation in B. subtilis and the signal transduction mechanism has a common feature in Gram-positive and Gram-negative bacteria.

Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp.

Peptide-synthetase-encoding DNA fragments were isolated by a PCR-based approach from the chromosome of Microcystis aeruginosa K-139, which produces cyclic heptapeptides, 7-desmethylmicrocystin-LR and 3,7-didesmethylmicrocystin-LR. Three open reading frames (mcyA, mcyB, mcyC) encoding microcystin synthetases were identified in the gene cluster. Sequence analysis indicated that McyA (315 kDa) consists of two modules with an N-methylation domain attached to the first and an epimerization domain attached to the second; McyB (242 kDa) has two modules, and McyC (147 kDa) contains one module with a putative C-terminal thioesterase domain. Conserved amino acid sequence motifs for ATP binding, ATP hydrolysis, adenylate formation, and 4'-phosphopantetheine attachment were identified by sequence comparison with authentic peptide synthetase. Insertion mutations in mcyA, generated by homologous recombination, abolished the production of both microcystins in M. aeruginosa K-139. Primer extension analysis demonstrated light-dependent mcy expression. Southern hybridization and partial DNA sequencing analyses of six microcystin-producing and two non-producing Microcystis strains suggested that the microcystin-producing strains contain the mcy gene and the non-producing strains can be divided into two groups, those possessing no mcy genes and those with mcy genes.

An intrinsic DNA curvature found in the cyanobacterium Microcystis aeruginosa K-81 affects the promoter activity of rpoD1 encoding a principal sigma factor.

The rpoD1 gene in the unicellular cyanobacterium Microcystis aeruginosa K-81 encodes a principal sigma factor of RNA polymerase and is transcribed under light and dark conditions to produce multiple monocistronic transcripts. In the 5'-upstream region from rpoD1 Promoter 2, which has a sequence of Escherichia coli type, we found a sequence-directed DNA curvature with an AT-rich sequence. Insertions of 2 to 21 base pairs introduced into the curved center changed a gross geometry of the original curved DNA structure. The rpoD1 promoter activities assayed in vivo by using transcriptional lacZ fusions were correlated with the change in the gross geometry in not only a cyanobacterium but also E. coli. In addition, RNA polymerase binding to the rpoD1 promoter region and the efficiency of the mRNA synthesis from the rpoD1 Promoter 2 were also affected in vitro by the change in the geometry. These results suggest that the tertiary structure of the curved DNA is important for the rpoD1 transcription. The deletion of the center region of the curvature resulted in a considerable reduction of the transcription from Promoter 2 in the cyanobacterium. This report demonstrates that a curved DNA plays a significant role in transcription in cyanobacteria, and that this functional curvature is located in the 5'-upstream region from the rpoD gene, which encodes a principal sigma factor in eubacteria.

Polyketide synthase gene coupled to the peptide synthetase module involved in the biosynthesis of the cyclic heptapeptide microcystin.

The peptide synthetase gene operon, which consists of mcyA, mcyB, and mcyC, for the activation and incorporation of the five amino acid constituents of microcystin has been identified [T. Nishizawa et al. (1999) J. Biochem. 126, 520-529]. By sequencing an additional 34 kb of DNA from microcystin-producing Microcystis aeruginosa K-139, we identified the residual microcystin synthetase gene operon, which consists of mcyD, mcyE, mcyF, and mcyG, in the opposite orientation to the mcyABC operon. McyD consisted of two polyketide synthase modules, and McyE contained a polyketide synthase module at the N-terminus and a peptide synthetase module at the C-terminus. McyF was found to exhibit similarity to amino acid racemase. McyG consisted of a peptide synthetase module at the N-terminus and a polyketide synthase at the C-terminus. The microcystin synthetase gene cluster was conserved in another microcystin-producing strain, Microcystis sp. S-70, which produces Microcystin-LR, -RR, and -YR. Insertional mutagenesis of mcyA, mcyD, or mcyE in Microcystis sp. S-70 abolished microcystin production. In conclusion, the mcyDEFG operon is presumed to be responsible for 3-amino-9-methoxy-2,6, 8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda) biosynthesis, and the incorporation of Adda and glutamic acid into the microcystin molecule.

The rpoD1 gene product is a principal sigma factor of RNA polymerase in Microcystis aeruginosa K-81.

We performed molecular characterization of the RpoD1 protein encoded by the rpoD1 gene isolated from a cyanobacterium, Microcystis aeruginosa K-81. The deduced amino acid sequence (416 aa, 48,871 Da) of RpoD1 exhibited extensive similarity to those of proteins of the eubacterial RpoD family (Escherichia coli sigma 70 homologs). We overproduced and purified RpoD1 (54 kDa) from E. coli. Biological and biochemical analyses suggested that RpoD1 has a function homologous to that of E. coli sigma 70 as follows: (i) the RpoD1 protein complemented an rpoD mutant of E. coli strain YN543 (rpoD285) and (ii) the heterologous RNA polymerase holoenzyme reconstituted from the E. coli core enzyme and recombinant RpoD1 was specifically transcribed from E. coli promoters. Furthermore, Western blot analysis with antiserum against Synechococcus sp. strain PCC 7942 RpoD1 (a principal sigma factor of the sigma 70 type) indicated that M. aeruginosa K-81 RpoD1 (sigma A1) is the principal sigma factor, which is a major component of the sigma subunit on exponential cell growth.

A novel bend of DNA CIT: changeable bending-center sites of an intrinsic curvature under temperature conditions.

We found a novel DNA curvature, which has changeable bending-center sites of an intrinsic curvature under temperature conditions (CIT) in the cyanobacterium strain Microcystis aeruginosa K-81. Circular permutation analyses (CPA) for CIT under different temperature conditions (4-50 degrees C) revealed that the changeable bending-center sites are located in the 5'-upstream region (-141 to -184) of the psbA2 gene, encoding the D1 protein homolog for photosynthesis. The nucleotide sequence around the bending center contains several dT (deoxy thymine) tracts, which seem to be a pivotal determinant for CIT.

Highly repetitive sequences and characteristics of genomic DNA in unicellular cyanobacterial strains.

Microcystis aeruginosa (Synechocystis) is a unicellular cyanobacterium that performs oxygenic photosynthesis. We found two novel sets of repetitive sequences, A (REP-A) and B (REP-B), on the M. aeruginosa K-81 genomic DNA, which consisted of distinct motifs of tandem repeated sequences located in the up- and downstream regions of the orf1 structural gene, respectively. Genomic Southern hybridization revealed multicopies of REP-A and -B on the genome. Furthermore, genomic Southern blots of cyanobacteria species with the REP-A and -B probes revealed that different hybridization signals appeared on the genomic DNAs of all 12 Microcystis strains, but no signal appeared on those of Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, and Anabaena sp. PCC 7120.

Restriction barrier composed of an extracellular nuclease and restriction endonuclease in the unicellular cyanobacterium Microcystis sp.

The unicellular cyanobacterium Microcystis aeruginosa K-81 has two types of restriction barrier, an extracellular nuclease and sequence-specific endonucleases. The nuclease was detected in the culture supernatant and it was easily released from the cells by washing with water or buffer containing Triton X-100. This nuclease was identified as a polypeptide of about 28 kDa that digested covalently closed circular and linear double-stranded DNAs, including chromosomal DNA from M. aeruginosa K-81. Among another 13 Microcystis strains examined, 3 produced an extracellular nuclease. Furthermore, M. aeruginosa K-81 contained two sequence-specific endonucleases, MaeK81I and MaeK81II, which were isoschizomers of SplI and Sau96I, respectively.

Quantitative analysis of the local effect of skin temperature on sweating.

Effects of local skin temperature on sweat gland activity were analyzed quantitatively by measuring changes in the rates of thermal sweating and of drug-induced sweating by local heating. The data indicates that a rise in local temperature causes an accelerated increase in the rate of sweat production, the Q10 being around 2.5 regardless of the basal sweat rate with some individual variations. Local heating apparently facilitates transmitter release at the neuroglandular junction and augments glandular responsiveness, their significances being comparable.

Effects of sweat gland training by repeated local heating.

Effects of sweat gland training by daily local heating were examined and its significance in heat acclimatization was evaluated. Training by 2-hr immersion of an arm in hot water of 43 degrees C caused distinct augmentation of sweat gland activity in the trained area, with reduction in the degree of hidromeiosis, when tested by an arm bag collection of sweat. Concentrations of sweat electrolytes also showed definite decreases. The general tendency that Na and Cl concentrations in sweat rise in the course of hidromeiosis was attenuated or even reversed after the training. The sweat test using resistance hygrometry failed to show a marked or consistent increase in sweat rate of the trained area, although an increase was the common case on the dorsum of the hand and the extensor aspect of the forearm. The effect of training appeared in a few days of training, developed progressively with training days and showed a tendency to develop even after 3 weeks of training. The same training in midsummer failed to exert significant effects on sweat gland activity, suggesting that the sweat gland had been naturally trained to a considerable degree by then. On the other hand, training by repeated radiant heating of a local area caused only obscure changes in the activity of sweat glands. The present results reveal that sweat glands can be trained to be resistant to hidromeiosis in a hot-humid environment and that such peripheral changes appear to play a predominant role in augmentation of sweating capacity in the early stage of heat acclimatization.

Occurrence of mental and thermal sweating on the human axilla.

Sweat responses to mental arithmetic were recorded simultaneously on the axilla, palm, and general body surface (chest and forearm) by resistance or capacitance hygrometry at different ambient temperatures. Sweat expulsions observed on the axilla were fully synchronized with those on the general body surface, but not always with those on the palm. In some subjects the sweat responses to mental arithmetic on the general body surface were different in pattern from those on the palm. In such subjects the sweat response pattern on the axilla was similar to that on the general body surface. The sweat response to mental arithmetic occurred at a considerably lower environmental temperature on the axilla than on the general body surface. The occurrence of the sweat response on the axilla can be related to the peculiar feature of axillary thermal sweating: a lower threshold temperature and less responsiveness to thermal load compared with thermal sweating on the general body surface. This suggests that mental sweating on the axilla occurs due to the characteristic feature of thermal sweating on the axilla. Axillary eccrine sweating is not different qualitatively from sweating on the general body surface.

Significance of skin pressure in body heat balance.

It has been demonstrated by Takagi and his colleagues that pressure on a specified area of the body surface causes depression of sweating in a certain body division and changes in the relative sweat rates between body divisions. Furthermore, skin pressure has been assumed to suppress the central thermoregulatory activity, thus bringing about a rise or fall in body temperature in a hot or cool environment, respectively. We examined the effect of skin pressure applied to the bilateral subaxillary regions on body heat balance by means of continuous recordings of evaporative weight loss (total sweat rate), local sweat rates at various areas and rectal and skin temperatures and measurements of metabolic rate. Most experiments were carried out at a room temperature of 36 degrees C with 40% rh and a few were done at 27 degrees C in the absence of thermal sweating. Various strengths of pressure up to 5 kg/50 cm2 were employed. It was observed that the total sweat rate was either unchanged, decreased or occasionally even increased. There was an apparent tendency that the stronger the pressure was, the more depressed was the total sweating. A weaker pressure, on the other hand, often caused facilitation of total sweating. Changes in rectal and mean body temperatures and in metabolic rate were minimal in the majority of cases, and bore no relationship to the changes in the total sweat rate. These results offer no evidence that skin pressure of up to 5 kg/50 cm2 affects human central thermoregulatory activity but suggest that it may exert a sweat-inhibitory effect, primarily through the interaction of sudomotor impulses somewhere along the efferent pathways, possibly at the spinal segmental level.